Antibodies to OX-2/CD200 and uses thereof

ABSTRACT

This application provides methods and compositions for modulating and/or depleting CD200 positive cells.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 ofInternational Application No. PCT/US2007/000711, filed Jan. 11, 2007,which claims the benefit of U.S. Provisional Application Nos.60/758,426, filed Jan. 12, 2006, 60/759,085, filed Jan. 12, 2006, and60/801,991, filed May 18, 2006, which applications are herebyincorporated by reference in their entireties. International ApplicationPCT/US2007/000711 was published under PCT Article 21(2) in English.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jul. 7, 2010, is namedALEXP10060.txt, and is 99,211 bytes in size.

TECHNICAL FIELD

The disclosure relates to OX-2/CD200 (herein referred to as CD200)antagonists and methods of depleting or eliminating cells overexpressingCD200 in a subject with cancer or autoimmune disease. The methods oftherapy for the treatment of cancer provide a combination of twomechanisms. More specifically, this disclosure relates to treatingcancer using a therapy that: (1) interferes with the interaction betweenCD200 and its receptor to block immune suppression thereby promotingeradication of the cancer cells; and/or (2) directly kills the cancercells either by (a) antibody-dependent cellular cytotoxicity orcomplement-mediated cytotoxicity or by (b) targeting cells using afusion molecule that includes a CD200-targeting portion. The disclosurealso relates to a method of treating autoimmune disorders by a therapythat increases the antibody-dependent cellular cytotoxicity and/orcomplement-mediated cytotoxicity of CD200-positive immune cells.

BACKGROUND

Various mechanisms play a role in the body's response to a diseasestate, including cancer. For example, CD⁺ T helper cells play a crucialrole in an effective immune response against various malignancies byproviding stimulatory factors to effector cells. Cytotoxic T cells arebelieved to be the most effective cells to eliminate cancer cells, and Thelper cells prime cytotoxic T cells by secreting Th1 cytokines such asIL-2 and IFN-γ. In various malignancies, T helper cells have been shownto have an altered phenotype compared to cells found in healthyindividuals. One of the prominent altered features is decreased Th1cytokine production and a shift to the production of Th2 cytokines.(See, e.g., Kiani, et al., Haematologica 88:754-761 (2003); Maggio, etal., Ann Oncol 13 Suppl 1:52-56 (2002); Ito, et al., Cancer 85:2359-2367(1999); Podhorecka, et al., Leuk Res 26:657-660 (2002); Tatsumi, et al.,J Exp Med 196:619-628 (2002); Agarwal, et al., Immunol Invest 32:17-30(2003); Smyth, et al., Ann Surg Oncol 10:455-462 (2003); Contasta, etal., Cancer Biother Radiopharm 18:549-557 (2003); Lauerova, et al.,Neoplasma 49:159-166 (2002).) Reversing that cytokine shift to a Th1profile has been demonstrated to augment anti-tumor effects of T cells.(See Winter, et al., Immunology 108:409-419 (2003); Inagawa, et al.,Anticancer Res 18:3957-3964 (1998).)

Mechanisms underlying the capacity of tumor cells to drive the cytokineexpression of T helper cells from Th1 to Th2 include the secretion ofcytokines such as IL-10 or TGF-β as well as the expression of surfacemolecules interacting with cells of the immune system. CD200, a moleculeexpressed on the surface of dendritic cells which possesses a highdegree of homology to molecules of the immunoglobulin gene family, hasbeen implicated in immune suppression (Gorczynski et al.,Transplantation 65:1106-1114 (1998)). It has been shown, for example,that CD200-expressing cells can inhibit the stimulation of Th1 cytokineproduction.

Although immune cells can help attack and eliminate cancer cells, incertain instances, such as in autoimmune disorders, allergies, and therejection of tissue or organ transplants, the immune system can be thecause of illness. In order to inhibit harmful immune reactions in suchinstances, immunosuppressive agents such as corticosteroids and cytokineantagonists may be administered to patients. However these generalimmunosuppressives can elicit undesirable side effects includingtoxicity and reduced resistance to infection. Thus alternative, andperhaps more specific, methods of treating autoimmunity are needed.

Several immunomodulatory therapies, including antibody therapies, haveproven successful in the treatment of certain cancers and autoimmunedisorders. However there is a clinical need for additional antibodytherapies for the treatment of both cancer and autoimmune disorders.Furthermore, there is a related need for humanized or other chimerichuman/mouse monoclonal antibodies. In well publicized studies, patientsadministered murine anti-TNF (tumor necrosis factor) monoclonalantibodies developed anti-murine antibody responses to the administeredantibody. (Exley A. R., et al., Lancet 335:1275-1277 (1990)). This typeof immune response to the treatment regimen, commonly referred to as thehuman anti-mouse antibody (HAMA) response (Mirick et al. Q J Nucl MedMol Imaging 2004; 48: 251-7), decreases the effectiveness of thetreatment and may even render the treatment completely ineffective.Humanized or chimeric human/mouse monoclonal antibodies have been shownto significantly decrease the HAMA response and to increase thetherapeutic effectiveness of antibody treatments. See, for example,LoBuglio et al., P.N.A.S. 86:4220-4224 (June 1989). Furthermore,antibodies in which particular functionalities are either enhanced orreduced may find useful applications in the clinic.

SUMMARY

This disclosure relates to agents and methods for modulating thefunction of CD200. Agents that modulate the function of CD200 includeagents that modulate the activity and/or expression of CD200 and/or itsreceptor (CD200R). In some embodiments, the agents inhibit the functionor activity of CD200. Thus in certain aspects, said agents act asantagonists to CD200. Certain antagonists may bind to CD200 and inhibitor disrupt the interaction of CD200 with its receptor. Other antagonistsmay bind to CD200 but may not block the CD200:CD200R interaction. ThusCD200 antagonists include any agent that is capable of modulating theeffects of CD200 by mechanisms that may or may not include blocking theCD200:CD200R interaction. CD200 antagonists include but are not limitedto polypeptides, small molecules, organometallic compounds,oligonucleotide constructs, RNAi constructs, aptamers, spiegelmers,antisense nucleic acids, locked nucleic acid (LNA) inhibitors, peptidenucleic acid (PNA) inhibitors, immunomodulatory agents, antibodies,antigen-binding fragments, prodrugs, and/or peptidomimetic compounds.

In certain embodiments, the said antagonist is an anti-CD200 antibody.Antibodies, as referred to herein, include antigen-binding fragments,Fab, Fv, scFv, Fab′ and F(ab′)₂, monoclonal and polyclonal antibodies,engineered antibodies (including chimeric, single chain, CDR-grafted,humanized, fully human antibodies, and artificially selectedantibodies), and synthetic or semi-synthetic antibodies.

In certain aspects, the present disclosure relates to chimeric,humanized, human and de-immunized anti-CD200 antibodies andantigen-binding fragments thereof. In further embodiments, an antibodyof the disclosure comprises a heavy chain comprising an amino acidsequence that is at least 90% identical to an amino acid sequenceselected from among SEQ ID NOS: 7, 9, 11, and 20, or fragments thereof.Included is an antibody comprising an amino acid sequence that is about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical orsimilar to an amino acid sequence provided in SEQ ID NOS: 7, 9, 11, and20, or fragments thereof (including but not limited to fragmentscorresponding to the sequences without the leader sequences). The saidantibody may additionally comprise a light chain comprising an aminoacid sequence that is at least about 90% identical or similar to anamino acid sequence selected from among SEQ ID NOS: 24, 26, 28, and 32,or fragments thereof. Likewise, the aforementioned amino acid sequencemay be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical or similar to an amino acid sequence provided in SEQ ID NOS:24, 26, 28, and 32, including fragments thereof (including but notlimited to fragments corresponding to the sequences without the leadersequences).

In one embodiment, the disclosure relates to an anti-CD200 antibodycomprising a heavy chain comprising an amino acid sequence that is atleast about 90% identical to the amino acid sequence of SEQ ID NO: 7 andalso comprising a light chain comprising an amino acid sequence that isat least about 90% identical to SEQ ID NO: 24. Also included areanti-CD200 antibodies comprising amino acid sequences that are about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical orsimilar to one or more amino acid sequence provided in SEQ ID NOS: 7 and24 or fragments thereof. Fragments include, but are not limited to,sequences corresponding to the sequences set forth in SEQ ID NOS: 7 and24 without the leader sequences. Accordingly, the disclosure relates toan anti-CD200 antibody comprising an amino acid sequence encoded by anucleic acid sequence that hybridizes under stringent conditions to thenucleic acid sequence of SEQ ID NO: 6 (including fragments thereof andcomplements thereto) and also comprising an amino acid sequence encodedby a nucleic acid sequence that hybridizes under stringent conditions tothe nucleic acid sequence of SEQ ID NO: 23 (including fragments thereofand complements thereto). Also included is an anti-CD200 antibodycomprising an amino acid sequence encoded by a nucleic acid sequencethat is at least about 80% homologous or similar to a nucleic acidsequence provided in SEQ ID NO: 6, including fragments thereof andcomplements thereto, and also comprising an amino acid sequence encodedby a nucleic acid sequence that is at least about 80% homologous orsimilar to a nucleic acid sequence provided in SEQ ID NO: 23, includingfragments thereof and complements thereto. The invention also relates toanti-CD200 antibodies comprising an amino acid sequence encoded by anucleic acid sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% homologous or similar to a nucleic acidsequence provided in SEQ ID NOS: 6 or 23, including fragments thereofand complements thereto.

In another embodiment, the disclosure relates to an anti-CD200 antibodycomprising a heavy chain comprising an amino acid sequence that is atleast about 90% identical to the amino acid sequence of SEQ ID NO: 9 andalso comprising an amino acid sequence that is at least about 90%identical to SEQ ID NO: 26. Also included are anti-CD200 antibodiescomprising amino acid sequences that are about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical or similar to one or moreamino acid sequence provided in SEQ ID NOS: 9 and 26 or fragmentsthereof. Fragments include, but are not limited to, sequencescorresponding to the sequences set forth in SEQ ID NOS: 9 and 26 withoutthe leader sequences. Accordingly, the disclosure relates to ananti-CD200 antibody comprising an amino acid sequence encoded by anucleic acid sequence that hybridizes under stringent conditions to thenucleic acid sequence of SEQ ID NO: 8 (including fragments thereof andcomplements thereto) and also comprising an amino acid sequence encodedby a nucleic acid sequence that hybridizes under stringent conditions tothe nucleic acid sequence of SEQ ID NO: 25 (including fragments thereofand complements thereto). Also included is an anti-CD200 antibodycomprising an amino acid sequence encoded by a nucleic acid sequencethat is at least about 80% homologous or similar to a nucleic acidsequence provided in SEQ ID NO: 8, including fragments thereof andcomplements thereto, and also comprising an amino acid sequence encodedby a nucleic acid sequence that is at least about 80% homologous orsimilar to a nucleic acid sequence provided in SEQ ID NO: 25, includingfragments thereof and complements thereto. The invention also relates toanti-CD200 antibodies comprising an amino acid sequence encoded by anucleic acid sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% homologous or similar to a nucleic acidsequence provided in SEQ ID NOS: 8 or 25, including fragments thereofand complements thereto.

In a further embodiment, the disclosure relates to an anti-CD200antibody comprising an amino acid sequence that is at least about 90%identical to the amino acid sequence of SEQ ID NO: 11 and alsocomprising an amino acid sequence that is at least about 90% identicalto SEQ ID NO: 26. Also included are anti-CD200 antibodies comprisingamino acid sequences that are about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical or similar to one or more amino acidsequence provided in SEQ ID NOS: 11 and 26 or fragments thereof.Fragments include, but are not limited to, sequences corresponding tothe sequences set forth in SEQ ID NOS: 11 and 26 without the leadersequences. Accordingly, the disclosure relates to an anti-CD200 antibodycomprising an amino acid sequence encoded by a nucleic acid sequencethat hybridizes under stringent conditions to the nucleic acid sequenceof SEQ ID NO: 10 (including fragments thereof and complements thereto)and also comprising an amino acid sequence encoded by a nucleic acidsequence that hybridizes under stringent conditions to the nucleic acidsequence of SEQ ID NO: 25 (including fragments thereof and complementsthereto). Also included is an anti-CD200 antibody comprising an aminoacid sequence encoded by a nucleic acid sequence that is at least about80% homologous or similar to a nucleic acid sequence provided in SEQ IDNO: 10, including fragments thereof and complements thereto, and alsocomprising an amino acid sequence encoded by a nucleic acid sequencethat is at least about 80% homologous or similar to a nucleic acidsequence provided in SEQ ID NO: 25, including fragments thereof andcomplements thereto. The invention also relates to anti-CD200 antibodiescomprising an amino acid sequence encoded by a nucleic acid sequencethat is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% homologous or similar to a nucleic acid sequence providedin SEQ ID NOS: 10 or 25, including fragments thereof and complementsthereto.

In an additional embodiment, the disclosure relates to an anti-CD200antibody comprising an amino acid sequence that is at least about 90%identical to the amino acid sequence of SEQ ID NO: 11 and alsocomprising an amino acid sequence that is at least about 90% identicalto SEQ ID NO: 28. Also included are anti-CD200 antibodies comprisingamino acid sequences that are about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical or similar to one or more amino acidsequence provided in SEQ ID NOS: 11 and 28 or fragments thereof.Fragments include, but are not limited to, sequences corresponding tothe sequences set forth in SEQ ID NOS: 11 and 28 without the leadersequences. Accordingly, the disclosure relates to an anti-CD200 antibodycomprising an amino acid sequence encoded by a nucleic acid sequencethat hybridizes under stringent conditions to the nucleic acid sequenceof SEQ ID NO: 10 (including fragments thereof and complements thereto)and also comprising an amino acid sequence encoded by a nucleic acidsequence that hybridizes under stringent conditions to the nucleic acidsequence of SEQ ID NO: 27 (including fragments thereof and complementsthereto). Also included is an anti-CD200 antibody comprising an aminoacid sequence encoded by a nucleic acid sequence that is at least about80% homologous or similar to a nucleic acid sequence provided in SEQ IDNO: 10, including fragments thereof and complements thereto, and alsocomprising an amino acid sequence encoded by a nucleic acid sequencethat is at least about 80% homologous or similar to a nucleic acidsequence provided in SEQ ID NO: 27, including fragments thereof andcomplements thereto. The invention also relates to anti-CD200 antibodiescomprising an amino acid sequence encoded by a nucleic acid sequencethat is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% homologous or similar to a nucleic acid sequence providedin SEQ ID NOS: 10 or 27, including fragments thereof and complementsthereto.

In yet another embodiment, the disclosure relates to an anti-CD200antibody, comprising an amino acid sequence that is at least about 90%identical to the amino acid sequence of SEQ ID NO: 20 and alsocomprising an amino acid sequence that is at least about 90% identicalto SEQ ID NO: 32. Also included are anti-CD200 antibodies comprisingamino acid sequences that are about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical or similar to one or more amino acidsequence provided in SEQ ID NOS: 20 and 32 or fragments thereof.Fragments include, but are not limited to, sequences corresponding tothe sequences set forth in SEQ ID NOS: 20 and 32 without the leadersequences. Accordingly, the disclosure relates to an anti-CD200 antibodycomprising an amino acid sequence encoded by a nucleic acid sequencethat hybridizes under stringent conditions to the nucleic acid sequenceof SEQ ID NO: 19 (including fragments thereof and complements thereto)and also comprising an amino acid sequence encoded by a nucleic acidsequence that hybridizes under stringent conditions to the nucleic acidsequence of SEQ ID NO: 31 (including fragments thereof and complementsthereto). Also included is an anti-CD200 antibody comprising an aminoacid sequence encoded by a nucleic acid sequence that is at least about80% homologous or similar to a nucleic acid sequence provided in SEQ IDNO: 19, including fragments thereof and complements thereto, and alsocomprising an amino acid sequence encoded by a nucleic acid sequencethat is at least about 80% homologous or similar to a nucleic acidsequence provided in SEQ ID NO: 31, including fragments thereof andcomplements thereto. Included, therefore, are anti-CD200 antibodiescomprising an amino acid sequence encoded by a nucleic acid sequencethat is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% homologous or similar to a nucleic acid sequence providedin SEQ ID NOS: 19 or 31, including fragments thereof and complementsthereto.

Anti-CD200 antibodies provided in the present disclosure includeantibodies and antigen-binding fragments with altered or no effectorfunction(s). Included are antibodies that comprise an altered constantor Fc region with either increased or decreased effector functions. Thedisclosure also relates to antibodies with altered or no effectorfunctions due to increased or decreased binding affinity, which mayarise from changes in the variable regions. Altered effector functionsinclude, for example, an increased or decreased ability to bind one ormore Fc receptor (FcR) or effector cell, increased or decreasedantigen-dependent cytotoxicity (ADCC), and/or increased or decreasedcomplement-dependent cytotoxicity (CDC). Variant antibodies include butare not limited to antibodies in which the constant region or Fc regioncontains one or more amino acid insertions, deletions, and/orsubstitutions. In additional embodiments, these variant antibodiescomprise a constant region wherein the CH1 and hinge region are derivedfrom human IgG2 and the CH2 and CH3 regions are derived from human IgG4.Also included are antibodies in which the constant or Fc region exhibitsaltered glycosylation. The aforementioned antibodies and antigen-bindingfragments (including single-chain antibodies) may be murine, chimeric,humanized, fully human, or de-immunized; included are antibodiescomprising the IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD, or IgEframeworks. Furthermore, the said antibodies, including fragments andvariants thereof, may be blocking or non-blocking antibodies orfragments thereof.

In certain aspects, the disclosure provides anti-CD200 antibodies thatexhibit decreased or no effector function. Antibodies with decreased orno effector function may comprise a variant or altered Fc or constantregion, such as, for example, a constant region with one or more aminoacid substitutions, insertions, and/or deletions, or a constant regionwith one or more changes in glycosylation. A variant constant regionincludes, for example, a region wherein one or more amino acids aresubstituted with alanine, such as in the Ala-Ala mutation describedherein, or wherein one or more carbohydrate groups is changed, added, orremoved. An alteration in the number and/or location of carbohydrategroups may be achieved by producing the said antibody in particular celltypes for which post-translational modifications would be reduced,absent, or increased. In one embodiment, effector function of anti-CD200antibodies is eliminated by swapping the IgG1 constant domain for anIgG2/4 fusion domain. Other ways of eliminating effector function can beenvisioned such as, e.g., mutation of the sites known to interact withan FcR or insertion of a peptide in the hinge region, therebyeliminating critical sites required for an FcR interaction.

In certain aspects and methods of the present disclosure, anti-CD200antibodies with altered or no effector functions comprise anti-CD200antibodies with one or more amino acid substitutions, insertions, and/ordeletions. In certain embodiments, such a variant anti-CD200 antibodyexhibits reduced or no effector function. In certain embodiments, thevariant constant region (of said variant antibody) possesses at leastabout 70% homology with the native sequence constant or Fc region and/orwith a constant or Fc region of the parent antibody or fragment thereof;in other embodiments the variant constant or Fc region possesses atleast about 80% homology or similarity therewith; in other embodimentsat least about 90% homology or similarity therewith and in additionalembodiments at least about 95% homology or similarity therewith. Inparticular embodiments, a variant antibody comprises a G2/G4 construct.Accordingly, the present disclosure relates to a constant or Fc regionof an anti-CD200 antibody with reduced or no effector function, whereinsaid constant region comprises a heavy chain comprising an amino acidsequence selected from the group consisting of SEQ ID NOS: 13, 15, 18,22, and fragments thereof. The present disclosure also relates tovariant constant regions of an anti-CD200 antibody wherein an antibodycomprises an amino acid sequence that is at least about 90% identical orsimilar to an amino acid sequence selected from among SEQ ID NOS: 13,15, 18, 22, and fragments thereof. Also included in the disclosure areantibodies comprising an amino acid sequence that is about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or similar to anamino acid sequence provided in SEQ ID NOS: 13, 15, 18, 22, andfragments thereof. Fragments include, but are not limited to, sequenceswithout the leader sequences. Additionally, in some embodiments aconstant region of an anti-CD200 antibody with reduced or no effectorfunction and comprising the G2/G4 construct is encoded by a nucleic acidselected from the group consisting of SEQ ID NOS: 12, 14, 16, 17, and21, or fragments thereof and complements thereto. In certainembodiments, an anti-CD200 antibody with reduced or no effector functionis encoded by a nucleic acid comprising a nucleic acid sequence that isat least about 80% homologous or similar to a sequence selected from SEQID NOS: 12, 14, 16, 17, and 21, including fragments thereof andcomplements thereto. In other embodiments, a variant anti-CD200 antibodyis encoded by a nucleic acid sequence comprising a sequence that isabout 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% homologous or similar to a nucleic acid sequence selected from thegroup consisting of SEQ ID NOS: 12, 14, 16, 17, and 21, includingfragments thereof and complements thereto. In still other embodiments,the nucleic acid encoding a variant anti-CD200 antibody comprises anucleic acid sequence that hybridizes under stringent conditions to anucleic acid sequence selected from the group consisting of SEQ ID NOS:12, 14, 16, 17, and 21, including fragments thereof and complementsthereto. Included are antigen-binding fragments and both blocking andnon-blocking antibodies or fragments thereof.

In one embodiment, the present disclosure relates to a variantanti-CD200 antibody comprising an amino acid sequence that is at leastabout 90% identical to the amino acid sequence of SEQ ID NO: 13 and alsocomprising an amino acid sequence that is at least about 90% identicalto SEQ ID NO: 28. Also included is an anti-CD200 antibody comprising oneor more amino acid sequence that is about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence providedin SEQ ID NOS: 13 and 28 or fragments thereof. Fragments include, butare not limited to, sequences corresponding to the sequences set forthin SEQ ID NOS: 13 and 28 without the leader sequences. Accordingly, thedisclosure relates to a variant anti-CD200 antibody comprising an aminoacid sequence encoded by a nucleic acid sequence that hybridizes understringent conditions to the nucleic acid sequence of SEQ ID NO: 12(including fragments thereof and complements thereto) and alsocomprising an amino acid sequence encoded by a nucleic acid sequencethat hybridizes under stringent conditions to the nucleic acid sequenceof SEQ ID NO: 27 (including fragments thereof and complements thereto).Also included is an anti-CD200 antibody comprising an amino acidsequence encoded by a nucleic acid sequence that is at least about 80%homologous to a nucleic acid sequence provided in SEQ ID NO: 12,including fragments thereof and complements thereto, and also comprisingan amino acid sequence encoded by a nucleic acid sequence that is atleast about 80% homologous to a nucleic acid sequence provided in SEQ IDNO: 27, including fragments thereof and complements thereto. Included,therefore, are anti-CD200 antibodies comprising an amino acid sequenceencoded by a nucleic acid sequence that is about 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to a nucleicacid sequence provided in SEQ ID NOS: 12 or 27, including fragmentsthereof and complements thereto.

In another embodiment, the disclosure relates to a variant anti-CD200antibody comprising an amino acid sequence that is at least about 90%identical to the amino acid sequence of SEQ ID NO: 15 and alsocomprising an amino acid sequence that is at least about 90% identicalto SEQ ID NO: 24. Also included is an anti-CD200 antibody comprising anamino acid sequence that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to one or more amino acid sequenceprovided in SEQ ID NOS: 15 and 24 or fragments thereof. Fragmentsinclude, but are not limited to, sequences corresponding to thesequences set forth in SEQ ID NOS: 15 and 24 without the leadersequences (e.g., the fragment of SEQ ID NO: 15 beginning at amino acid20 or 21). Accordingly, the disclosure relates to a variant anti-CD200antibody comprising an amino acid sequence encoded by a nucleic acidsequence that hybridizes under stringent conditions to the nucleic acidsequence of SEQ ID NO: 14 (including fragments thereof and complementsthereto) and also comprising an amino acid sequence encoded by a nucleicacid sequence that hybridizes under stringent conditions to the nucleicacid sequence of SEQ ID NO: 23 (including fragments thereof andcomplements thereto). Also included is an anti-CD200 antibody comprisingan amino acid sequence encoded by a nucleic acid sequence that is atleast about 80% homologous to a nucleic acid sequence provided in SEQ IDNO: 14, including fragments thereof and complements thereto, and alsocomprising an amino acid sequence encoded by a nucleic acid sequencethat is at least about 80% homologous to a nucleic acid sequenceprovided in SEQ ID NO: 23, including fragments thereof and complementsthereto. Included, therefore, are anti-CD200 antibodies comprising anamino acid sequence encoded by a nucleic acid sequence that is about80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%homologous to a nucleic acid sequence provided in SEQ ID NOS: 14 or 23,including fragments thereof and complements thereto.

In an additional embodiment, the disclosure relates to a variantanti-CD200 antibody comprising an amino acid sequence that is at leastabout 90% identical to the amino acid sequence of SEQ ID NO: 13 and alsocomprising an amino acid sequence that is at least 90% identical to SEQID NO: 28. Also included is an anti-CD200 antibody comprising one ormore amino acid sequence that is about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence providedin SEQ ID NOS: 13 and 28 or fragments thereof. Fragments include, butare not limited to, sequences corresponding to the sequences set forthin SEQ ID NOS: 13 and 28 without the leader sequences. Accordingly, thedisclosure relates to a variant anti-CD200 antibody comprising an aminoacid sequence encoded by a nucleic acid sequence that hybridizes understringent conditions to the nucleic acid sequence of SEQ ID NO: 16(including fragments thereof and complements thereto) and alsocomprising an amino acid sequence encoded by a nucleic acid sequencethat hybridizes under stringent conditions to the nucleic acid sequenceof SEQ ID NO: 27 (including fragments thereof and complements thereto).Also included is an anti-CD200 antibody comprising an amino acidsequence encoded by a nucleic acid sequence that is at least about 80%homologous to a nucleic acid sequence provided in SEQ ID NO: 16,including fragments thereof and complements thereto, and also comprisingan amino acid sequence encoded by a nucleic acid sequence that is atleast about 80% homologous to a nucleic acid sequence provided in SEQ IDNO: 27, including fragments thereof and complements thereto. Included,therefore, are anti-CD200 antibodies comprising an amino acid sequenceencoded by a nucleic acid sequence that is about 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to a nucleicacid sequence provided in SEQ ID NOS: 16 or 27, including fragmentsthereof and complements thereto.

In still another embodiment, the disclosure relates to a variantanti-CD200 antibody comprising an amino acid sequence that is at leastabout 90% identical to the amino acid sequence of SEQ ID NO: 18 and alsocomprising an amino acid sequence that is at least about 90% identicalto SEQ ID NO: 30. Also included is an anti-CD200 antibody comprising anamino acid sequence that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to an amino acid sequence provided inSEQ ID NOS: 18 and 30 or fragments thereof. Accordingly, the disclosurerelates to a variant anti-CD200 antibody comprising an amino acidsequence encoded by a nucleic acid sequence that hybridizes understringent conditions to the nucleic acid sequence of SEQ ID NO: 17(including fragments thereof and complements thereto) and alsocomprising an amino acid sequence encoded by a nucleic acid sequencethat hybridizes under stringent conditions to the nucleic acid sequenceof SEQ ID NO: 29 (including fragments thereof and complements thereto).Also included is an anti-CD200 antibody comprising an amino acidsequence encoded by a nucleic acid sequence that is at least about 80%homologous to a nucleic acid sequence provided in SEQ ID NO: 17,including fragments thereof and complements thereto, and also comprisingan amino acid sequence encoded by a nucleic acid sequence that is atleast 80% homologous to a nucleic acid sequence provided in SEQ ID NO:29, including fragments thereof and complements thereto. Included,therefore, are anti-CD200 antibodies comprising an amino acid sequenceencoded by a nucleic acid sequence that is about 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to a nucleicacid sequence provided in SEQ ID NOS: 17 or 29, including fragmentsthereof and complements thereto.

In another embodiment, the disclosure relates to a variant anti-CD200antibody comprising an amino acid sequence that is at least about 90%identical to the amino acid sequence of SEQ ID NO: 22 and alsocomprising an amino acid sequence that is at least about 90% identicalto SEQ ID NO. 34: Also included is an anti-CD200 antibody comprising anamino acid sequence that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to an amino acid sequences provided inSEQ ID NOS: 22 and 34 or fragments thereof. Accordingly, the disclosurerelates to a variant anti-CD200 antibody comprising an amino acidsequence encoded by a nucleic acid sequence that hybridizes understringent conditions to the nucleic acid sequence of SEQ ID NO: 21(including fragments thereof and complements thereto) and alsocomprising an amino acid sequence encoded by a nucleic acid sequencethat hybridizes under stringent conditions to the nucleic acid sequenceof SEQ ID NO: 33 (including fragments thereof and complements thereto).Also included is an anti-CD200 antibody comprising an amino acidsequence encoded by a nucleic acid sequence that is at least about 80%homologous to a nucleic acid sequence provided in SEQ ID NO: 21,including fragments thereof and complements thereto, and also comprisingan amino acid sequence encoded by a nucleic acid sequence that is atleast about 80% homologous to a nucleic acid sequence provided in SEQ IDNO: 33, including fragments thereof and complements thereto. Included,therefore, are anti-CD200 antibodies comprising an amino acid sequenceencoded by a nucleic acid sequence that is about 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to a nucleicacid sequence provided in SEQ ID NOS: 21 and 33, including fragmentsthereof and complements thereto.

Anti-CD200 antibodies with altered effector function may also exhibitincreased effector function. Increased effector functions include butare not limited to increased binding to one or more Fc receptors,increased ability to elicit ADCC, and/or increased ability to elicitCDC. Anti-CD200 antibodies with increased effector function may alsocomprise a variant Fc or constant region as described herein. Theaforementioned anti-CD200 antibodies with altered effector functions mayfurthermore be blocking or non-blocking antibodies. For example, ananti-CD200 antibody with increased effector function may bind to CD200but may not block the CD200:CD200R interaction. Such an antibody may beuseful when targeting an effector function (e.g., ADCC or CDC) to aCD200-expressing cell. As mentioned previously, antibodies describedherein, including the aforementioned anti-CD200 antibodies with alteredeffector function(s), include murine, chimeric, humanized, fully humanand de-immunized antibodies, all in their blocking and non-blockingforms, and fragments thereof.

In certain aspects, this disclosure provides methods and compositionsfor modulating or depleting CD200-positive cells. CD200-positive cellsmay be modulated or depleted by administering a CD200 antagonist to asubject. The said antagonist may target CD200-positive cells foreffector function and/or may disrupt the CD200:CD200R interaction. Incertain embodiments, the said antagonist is an anti-CD200 antibody. Thesaid anti-CD200 antibody may be an antibody described herein, includingany fragments and variants thereof. Included are antibodies andantigen-binding fragments with altered effector function(s), such as,for example, anti-CD200 antibodies with decreased or no effectorfunction. Also included are murine, chimeric, humanized, fully human andde-immunized antibodies and antigen-binding fragments, includingsingle-chain antibodies. The aforementioned antibodies may benon-blocking or blocking antibodies and include antibodies comprisingthe IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA, IgD, or IgEframeworks.

CD200-positive cells are implicated in certain types of cancers andcertain autoimmune diseases. Accordingly, CD200-positive cells includebut are not limited to immune cells (such as, e.g., B-cells and T-cells)and cancer cells (such as, e.g., cancer cells of ovarian, skin, lung,renal, breast, prostate, neuroblastoma, lymphoma, myeloma, leukemia,thyroid, and plasma cell cancers). Also included are cancer cells fromany tissue or organ derived from neural crest cells. Thus the subject inneed of a method of modulating or depleting CD200-positive cells may bea patient with cancer or autoimmune disease, or a patient who hasreceived or is expected to receive an organ transplant.

In one aspect this disclosure provides methods and compositions fortreating autoimmune disease. Autoimmune diseases that may be treated bythe methods and compositions provided herein include but are not limitedto rheumatoid arthritis, inflammatory bowel disease (includingulcerative colitis and Crohn's disease), systemic lupus erythematosus,multiple sclerosis, Hashimoto's thyroiditis, pernicious anemia,Addison's disease, type I diabetes, dermatomyositis, Sjogren's syndrome,lupus erythematosus, myasthenia gravis, Reiter's syndrome, Grave'sdisease, psoriasis, and autoimmune hemolytic diseases. In someembodiments, a patient with autoimmune disease is given an antagonist toCD200, and in certain embodiments, the antagonist is an anti-CD200antibody. The anti-CD200 antibody may comprise a variant constant regionas described herein. Accordingly, the anti-CD200 antibody may exhibitaltered effector function(s), such as, for example, increased effectorfunction(s). The said antibody may exhibit, for example, increasedbinding to one or more Fc receptors. Additionally, the said antibody mayelicit increased ADCC and/or CDC. The said antibody may furthermore beeither a blocking or non-blocking antibody or fragment thereof and maybe either a murine, chimeric, humanized, fully human or de-immunizedantibody or fragment thereof.

Cancers for which the disclosed methods may be used include but are notlimited to melanoma, ovarian cancer, renal cancer, neuroblastoma, lungcancer, breast cancer, prostate cancer, lymphoma, myeloma, leukemia, andplasma cell cancers. Also included are cancers derived from neural crestcells and any cancers that express CD200. In certain embodiments, thisdisclosure provides a method for treating hematological malignancies,such as, for example, leukemias including chronic lymphocytic leukemia.

In a particularly useful embodiment, a cancer therapy in accordance withthis disclosure comprises (1) administering an anti-CD200 antibody orantagonist that interferes with the interaction between CD200 and itsreceptor to block immune suppression, thereby promoting eradication ofthe cancer cells; and/or (2) administering a fusion molecule thatincludes a CD-200 targeting portion to directly kill cancer cells.Alternatively, the antibody directly kills cancer cells throughcomplement-mediated and/or antibody-dependent cellular cytotoxicity. Invarious embodiments, the effector function of the anti-CD200 antibody isaltered. In one particular embodiment, the anti-CD200 antibody containsa variant or altered constant region for which the effector function isdecreased or eliminated; such an antibody may be useful for the methodsdescribed above in (1) and (2), for example.

In certain embodiments, the disclosure relates to fusion moleculeswherein an anti-CD200 antibody or antigen-binding fragment is linked toa second molecule. The said fusion molecule may comprise, for example, asmall molecule, polypeptide, peptidomimetic, heteroclitic peptide, achemotherapeutic agent, an immunomodulatory agent, a targeting moiety,or a nucleic acid construct (e.g., antisense, RNAi, or gene-targetingconstruct). The disclosure also includes antigen-binding fragments toCD200 wherein the fragment is fused or otherwise linked to apolypeptide, protein domain, serum protein, albumin, PEG (polyethyleneglycol), or any other molecule that will increase the half-life of thesaid fragment in vivo. Said antigen-binding fragments include Fab, Fv,single-chain fragments or scFv, Fab′, and F(ab′)₂, for example.

The present disclosure also relates to methods employing anti-CD200antibodies to determine the CD200 expression status of a cell or tissuesample obtained from a patient. Such methods include but are not limitedto immunohistochemical staining of tissue samples and flow cytometryanalysis of CD200-stained cells from a patient. The patient may be apatient with cancer, for example.

In accordance with the methods and compositions described herein, thedisclosure also relates to methods of treating a transplant or allograftpatient. An anti-CD200 antibody or other CD200 antagonist of the presentdisclosure may be administered to a patient prior to a transplant orallograft procedure or after the procedure in order to decrease oreliminate CD200-positive immune cells that could reduce the patient'sacceptance of the transplanted organ or tissue. In a particularembodiment, an anti-CD200 antibody with increased effector function isgiven to a transplant patient.

In further embodiments, methods are provided for combination therapiescomprising anti-CD200 therapy. For example, a patient receiving a firsttherapy comprising a CD200 antagonist (e.g., an anti-CD200 antibodydescribed herein) may also be given a second therapy. The CD200antagonist may be given simultaneously with the second therapy.Alternatively, the CD200 antagonist may be given prior to or followingthe second therapy. Second therapies include but are not limited tochemotherapeutic agents, radiation therapy, vaccines, antibiotics andanti-viral agents, and immunomodulatory therapies.

In another embodiment of the present disclosure, methods are providedfor monitoring the progress of a therapeutic treatment. The methodinvolves administering a therapy (e.g. an immunomodulatory therapy, achemotherapeutic therapy, etc.) and determining CD200 levels in asubject at least twice to determine the effectiveness of the therapy.Other methods to determine the effectiveness of a therapy include butare not limited to detection of cancer cells, total lymphocyte count,spleen, liver, and/or lymph node size, number of regulatory T cells,intracellular or serum cytokine profiles, or secretion of cytokines by Tor B cells as measured by ELISPOT—an assay system that allows thedetection of cytokines or other secreted molecules on a per cell basis.

According to the compositions and methods set forth in the presentembodiments, the disclosure also relates to any pharmaceuticalcomposition comprising an anti-CD200 antibody. Included are chimeric,humanized, human and de-immunized anti-CD200 antibodies andantigen-binding fragments, including single-chain antibodies. Alsoincluded are murine, chimeric, humanized, human and de-immunized variantanti-CD200 antibodies and antigen-binding fragments with alteredeffector function(s) as described herein. The aforementioned antibodiesand variant antibodies may either be blocking or non-blocking antibodiesor antigen-binding fragments.

In certain embodiments, patients for whom anti-CD200 therapy is usefulor patients who expect to receive a therapy comprising a CD200antagonist therapy (including, for example, an anti-CD200 antibody) maybe screened for certain previously received treatments and procedures orfor current medical status. In one embodiment for example, femalepatients may be pre-screened for pregnancy and agree to contraception,since CD200 plays an important role in protection against abortion.Accordingly, patients receiving said therapy may agree to practice oneor more methods of contraception. The said patient may agree to use oneor more methods of contraception for a designated period prior tostarting the said therapy and/or for the duration of the said therapy.In certain embodiments, female patients receive counseling concerningthe risks with respect to fetal exposure to such anti-CD200 therapy. Inadditional embodiments, such patients may be expected to sign informedconsent forms prior to such treatment. In other aspects, physicalscounseling patients regarding anti-CD200 therapy may require suchpatients to use contraceptive devices or formulations prior toadministering the anti-CD200 therapy (see, for example, U.S. Pat. No.6,908,432 and related patents, the contents of which are incorporatedherein by reference). Similarly, in other embodiments, patients may bescreened to identify patients who have previously received brain surgeryand/or radiation therapy to the brain; anti-CD200 therapy would not beprescribed for such patients.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the nucleic acid sequence for the primer C7 mhHF (SEQ IDNO:1) used in generating the G2/G4 construct.

FIG. 2 provides the nucleic acid sequence for the Primer Rev Age Pri(SEQ ID NO: 2) used in generating the G2/G4 construct.

FIG. 3 provides the nucleic acid sequence for the primer C2aB7 rev (SEQID NO: 3) used in generating the G2/G4 construct.

FIG. 4 provides the nucleic acid sequence for the lacpri (SEQ ID NO: 4)used in generating the G2/G4 construct.

FIG. 5 provides the nucleic acid sequence for LeadVHpAX (SEQ ID NO: 5).

FIGS. 6A-F depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody chC2aB7-hG1 (SEQ ID NOS: 6, 7,23, 24, 37, and 38). FIG. 6C shows SEQ ID NO: 37 (nucleic acid sequence)and SEQ ID NO: 7 (amino acid sequence). SEQ ID NO: 7 as shown in theschematic is contiguous but is depicted with a corresponding nucleotidesequence that includes introns. FIG. 6F shows SEQ ID NO: 38 (nucleicacid sequence) and SEQ ID NO: 24 (amino acid sequence).

FIGS. 7A-F depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody hB7V4V1-hG1(SEQ ID NOS: 8, 9, 25,26, 39, and 40). FIG. 7C shows SEQ ID NO: 39 (nucleic acid sequence) andSEQ ID NO: 9 (amino acid sequence). FIG. 7F shows SEQ ID NO: 40 (nucleicacid sequence) and SEQ ID NO: 26 (amino acid sequence).

FIGS. 8A-F depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody hB7V3V1-hG1 (SEQ ID NOS: 10, 11,25, 26, 40, and 41). FIG. 8C shows SEQ ID NO: 41 (nucleic acid sequence)and SEQ ID NO: 11 (amino acid sequence). FIG. 8F shows SEQ ID NO: 41(nucleic acid sequence) and SEQ ID NO: 26 (amino acid sequence).

FIGS. 9A-F depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody hB7V3V2-hG1(SEQ ID NOS: 10, 11,27, 28, 41, and 42). FIG. 9C shows SEQ ID NO: 41 (nucleic acid sequence)and SEQ ID NO: 11 (amino acid sequence). FIG. 9F shows SEQ ID NO: 42(nucleic acid sequence) and SEQ ID NO: 28 (amino acid sequence).

FIGS. 10A-F depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody hB7V3V2-hG2G4 (SEQ ID NOS: 12,13, 27, 28, 42, and 43). FIG. 10C shows SEQ ID NO: 43 (nucleic acidsequence) and SEQ ID NO: 13 (amino acid sequence). SEQ ID NO: 13 asshown in the schematic is contiguous but is depicted with acorresponding nucleotide sequence that includes introns. FIG. 10F showsSEQ ID NO: 42 (nucleic acid sequence) and SEQ ID NO: 28 (amino acidsequence).

FIGS. 11A-F depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody chC2aB7-hG2G4 (SEQ ID NOS: 14,15, 23, 24, 44, 45, 46, and 47). FIG. 11C shows SEQ ID NO: 44 (nucleicacid sequence) and SEQ ID NO: 45 (amino acid sequence). SEQ ID NO: 45corresponds to amino acids 1-337 of SEQ ID NO: 15. As shown in theschematic, SEQ ID NO: 45 is contiguous but is depicted with acorresponding nucleotide sequence that includes introns. FIG. 11F showsSEQ ID NO: 46 (nucleic acid sequence) and SEQ ID NO: 47 (amino acidsequence).

FIGS. 12A-F depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody hB7V3V2-cG2G4 (SEQ ID NOS: 13,16, 27, 28, 48, and 49). FIG. 12C shows SEQ ID NO: 48 (nucleic acidsequence) and SEQ ID NO: 13 (amino acid sequence). FIG. 12F shows SEQ IDNO: 49 (nucleic acid sequence) and SEQ ID NO: 28 (amino acid sequence).

FIGS. 13A-D depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody chC7-hG2G4 (SEQ ID NOS: 17, 18,29, and 30).

FIGS. 14A-F depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody D1B5-hG1 (SEQ ID NOS: 19, 20, 31,32, 50, and 51). FIG. 14C shows SEQ ID NO: 50 (nucleic acid sequence)and SEQ ID NO: 20 (amino acid sequence). SEQ ID NO: 20 as shown in theschematic is contiguous but is depicted with a corresponding nucleotidesequence that includes introns. FIG. 14F shows SEQ ID NO: 51 (nucleicacid sequence) and SEQ ID NO: 32 (amino acid sequence).

FIGS. 15A-D depict the amino acid sequences and nucleotide sequences forthe heavy and light chains of antibody G2G4 63L1D (SEQ ID NOS: 21, 22,33, and 34).

FIG. 16 provides the nucleic acid sequence for the forward primer forcloning CD200 cDNA (SEQ ID NO: 35).

FIG. 17 provides the nucleic acid sequence for the reverse primer forcloning CD200 cDNA (SEQ ID NO: 36).

FIG. 18 shows the effects of administering humanized CD200 antibodies inthe RAJI-CD200/PBL model. Humanized anti-CD200 antibodies resulted in aninhibition of tumor growth.

FIG. 19 demonstrates the effects of administering humanized CD200antibodies with and without effector function in the Namalwa_CD200animal model. Antibodies without effector function exhibited efficacy ininhibiting tumor growth.

FIG. 20 is a table showing the expression level of CD200 in chroniclymphocytic leukemia (CLL) patient samples compared to normal samples.

FIG. 21 depicts the relative levels of CD200 expression detected incancer cell lines.

FIG. 22 shows the expression level of CD200 antigen in human ovariancancer samples relative to the expression level detected in humanperipheral blood lymphocytes (PBL).

FIG. 23 shows the expression level of CD200 antigen in human melanomapatient samples relative to the expression level detected in PBL.

FIG. 24 shows immunohistochemical staining of CD200 of melanoma patientsamples.

FIG. 25 demonstrates the effects of anti-CD200 antibody in cytokineproduction. The levels of IL-2 production in mixed cell populationassays were measured in the absence and presence of CD200 antibody. Theantibody used is a chimeric anti-CD200 antibody with no effectorfunction.

FIG. 26 shows the effects of administering anti-CD200 antibodies, withor without effector function, in the Namalwa/PBL model in which thetumors do not express CD200.

FIG. 27 shows flow cytometric analysis of CD200 expression on activatedT-cells. CD3+ cells were activated with mOKT3, harvested, washed andsubjected to staining with the indicated conjugated antibodies specificfor human CD25, CD200, CD5, CD4 and CD8. Cells were washed and analyzedfor immunofluorescence on a FacsCaliber flow cytometer using CellQuestsoftware.

FIG. 28 demonstrates the effects of anti-CD200 antibodies on ADCC ofactivated T-cells. CD3+ human T cells were stimulated with 10 μg/mLimmobilized (plate-coated) mOKT3 for 72 hrs. Activated T cells were thenchromated for use as targets and incubated with purified autologousCD56+ (NK) cells as effector cells. Cells were coincubated for 4 hoursat 37° C. at 25:1 (A) or 10:1 (B) effector: target cell ratios in thepresence or absence of a humanized anti-CD200 antibody capable ofmediating effector function (V3V2-G1) or engineered to lack effectorfunction (V3V2-G2G4). Data is represented as percent specific lysis.Anti-CD200 antibody increased ADCC of activated T-cells, whereas theanti-CD200 antibody with no effector function failed to induce ADCC.

FIG. 29 is a table showing the expression level of CD200 on plasmacells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

I. CD200 Antagonists

CD200 is a highly conserved type I transmembrane glycoprotein expressedon various cell types including cells of the immune system (e.g.,T-cells, B-cells, and dendritic cells (Barclay et al., TRENDS Immunol.2002: 23)) as well as certain cancer cells as shown herein. The proteininteracts with its receptor CD200R on myeloid cells and sub-populationsof T cells (Wright et al. J. Immunol. 2003 (171): 3034-3046 and Wrightet al., Immunity 2000 (13):233-242); the CD200:CD200R interactiondelivers an immunomodulatory signal to cells and inducesimmunosuppression including apoptosis-associated immune tolerance(Rosenblum et al. 2004 Blood (103): 2691-2698). Thus agents thatinterfere with the function or activity of CD200 and/or its receptor mayinhibit or reverse the immunosuppressive effects of the CD200:CD200Rinteraction. In addition, agents that specifically bind CD200 (but thatmay or may not inhibit the CD200:CD200R interaction) may triggerdownstream events that reverse or abolish the effects of CD200.

In certain aspects, the present disclosure relates to CD200 antagonists.As used herein, the term antagonist includes any agent that is capableof inhibiting the activity, function and/or the expression of CD200 orits receptor. Examples include but are not limited to polypeptides,antibodies, small molecules, aptamers, spiegelmers, locked nucleic acid(LNA) inhibitors, peptide nucleic acid (PNA) inhibitors, nucleic acidconstructs (e.g., gene-targeting constructs, antisense constructs, RNAinterference (RNAi) constructs, etc.) and peptidomimetics. In certainembodiments, the antagonist disrupts the interaction of CD200 andCD200R. In other embodiments, the CD200 antagonists are capable ofdecreasing the immunosuppressive effects of CD200 or are capable oftargeting CD200-expressing cells for depletion or elimination.

In certain aspects, the CD200 antagonists are polypeptides. Polypeptidesutilized in the present disclosure can be constructed using differenttechniques which are known to those skilled in the art. In oneembodiment, the polypeptides are obtained by chemical synthesis. Inother embodiments, the polypeptides are antibodies constructed from afragment or several fragments of one or more antibodies. In furtherembodiments, the polypeptide is an anti-CD200 antibody as describedherein.

Thus in certain embodiments, the CD200 antagonists are anti-CD200antibodies. As used herein, the term “antibodies” refers to completeantibodies or antibody fragments capable of binding to CD200 or CD200R.Included are Fab, Fv, scFv, Fab′ and F(ab′)₂, monoclonal and polyclonalantibodies, engineered antibodies (including chimeric, single chain,CDR-grafted, humanized, fully human antibodies, and artificiallyselected antibodies), and synthetic or semi-synthetic antibodiesproduced using phage display or alternative techniques. Also includedare antibodies engineered or produced in ways to contain variant oraltered constant or Fc regions with either increased or decreasedability to bind one or more effector cell; such variant antibodiesinclude but are not limited to antibodies in which the constant or Fcregion contains altered glycosylation patterns. Small fragments, such asFv and scFv, possess advantageous properties for diagnostic andtherapeutic applications on account of their small size and consequentsuperior tissue distribution. Antibodies with engineered or variantconstant or Fc regions can be useful in modulating effector functions,such as, for example, ADCC and CDC. Such antibodies with engineered orvariant constant or Fc regions may be useful in instances where CD200 isexpressed in normal tissue, for example; variant anti-CD200 antibodieswithout effector function in these instances may elicit the desiredtherapeutic response while not damaging normal tissue. Furthermore,antibodies, variant antibodies, and fragments thereof may be blocking(i.e., the said antibodies or fragments inhibit the interaction of CD200and CD200R) or non-blocking (i.e., the said antibodies or fragments bindto CD200 but do not block its interaction with CD200R).

The disclosure also relates to anti-CD200 antibodies comprising heavyand light chains as provided herein, including heavy and light chainsthat are homologous or similar to the heavy and/or light chains providedherein. “Homology” or “identity” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules.Homology and identity can each be determined by comparing a position ineach sequence which may be aligned for purposes of comparison. When anequivalent position in the compared sequences is occupied by the samebase or amino acid, then the molecules are identical at that position;when the equivalent site occupied by the same or a similar amino acidresidue (e.g., similar in steric and/or electronic nature), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of identical or similar amino acids atpositions shared by the compared sequences. The term “homology”describes a mathematically based comparison of sequence similaritieswhich is used to identify genes or proteins with similar functions ormotifs. As used herein, “identity” means the percentage of identicalnucleotide or amino acid residues at corresponding positions in two ormore sequences when the sequences are aligned to maximize sequencematching, i.e., taking into account gaps and insertions. Thus methods todetermine identity are designed to give the largest match between thesequences tested (see Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 1988, Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984), BLASTP, BLASTN, FASTA (Altschul, S. F. et al., J. Molec. Biol.215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402(1997)) and BLAST X (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410(1990). A sequence which is “unrelated” or “non-homologous” shares lessthan 40% identity, though preferably less than 25% identity with asequence of the present disclosure. In comparing two sequences, theabsence of residues (amino acids or nucleic acids) or presence of extraresidues also decreases the identity and homology/similarity.

Accordingly, the disclosure relates to antibodies as described hereinwithout the leader sequences. Thus antibodies of the disclosure maycomprise heavy and light chains (as described herein) in which theleader sequence is either absent or replaced by a different leadersequence. If host cells are used to produce antibodies of the presentdisclosure, appropriate leader sequences may therefore be selectedaccording to the particular host cell used.

Antibodies may be produced by methods well known in the art. Forexample, monoclonal anti-CD200 antibodies may be generated using CD200positive cells, CD200 polypeptide, or a fragment of CD200 polypeptide,as an immunogen, thus raising an immune response in animals from whichantibody-producing cells and in turn monoclonal antibodies may beisolated. The sequence of such antibodies may be determined and theantibodies or variants thereof produced by recombinant techniques.Recombinant techniques may be used to produce chimeric, CDR-grafted,humanized and fully human antibodies based on the sequence of themonoclonal antibodies as well as polypeptides capable of binding toCD200.

Moreover, antibodies derived from recombinant libraries (“phageantibodies”) may be selected using CD200-positive cells, or polypeptidesderived therefrom, as bait to isolate the antibodies or polypeptides onthe basis of target specificity. The production and isolation ofnon-human and chimeric anti-CD200 antibodies are well within the purviewof the skilled artisan.

Recombinant DNA technology is used to improve the antibodies produced innon-human cells. Thus, chimeric antibodies may be constructed in orderto decrease the immunogenicity thereof in diagnostic or therapeuticapplications. Moreover, immunogenicity may be minimized by humanizingthe antibodies by CDR grafting and, optionally, framework modification.See, U.S. Pat. No. 5,225,539, the contents of which are incorporatedherein by reference.

Antibodies may be obtained from animal serum or, in the case ofmonoclonal antibodies or fragments thereof, produced in cell culture.Recombinant DNA technology may be used to produce the antibodiesaccording to established procedure, including procedures in bacterial orpreferably mammalian cell culture. The selected cell culture systempreferably secretes the antibody product.

In another embodiment, a process for the production of an antibodydisclosed herein includes culturing a host, e.g. E. coli or a mammaliancell, which has been transformed with a hybrid vector. The vectorincludes one or more expression cassettes containing a promoter operablylinked to a first DNA sequence encoding a signal peptide linked in theproper reading frame to a second DNA sequence encoding the antibodyprotein. The antibody protein is then collected and isolated.Optionally, the expression cassette may include a promoter operablylinked to polycistronic, for example bicistronic, DNA sequences encodingantibody proteins each individually operably linked to a signal peptidein the proper reading frame.

Multiplication of hybridoma cells or mammalian host cells in vitro iscarried out in suitable culture media, which include the customarystandard culture media (such as, for example Dulbecco's Modified EagleMedium (DMEM) or RPMI 1640 medium), optionally replenished by amammalian serum (e.g. fetal calf serum), or trace elements and growthsustaining supplements (e.g. feeder cells such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages,2-aminoethanol, insulin, transferrin, low density lipoprotein, oleicacid, or the like). Multiplication of host cells which are bacterialcells or yeast cells is likewise carried out in suitable culture mediaknown in the art. For example, for bacteria suitable culture mediainclude medium LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, orM9 Minimal Medium. For yeast, suitable culture media include medium YPD,YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations andallows scale-up production to give large amounts of the desiredantibodies. Techniques for bacterial cell, yeast, plant, or mammaliancell cultivation are known in the art and include homogeneous suspensionculture (e.g. in an airlift reactor or in a continuous stirrer reactor),and immobilized or entrapped cell culture (e.g. in hollow fibres,microcapsules, on agarose microbeads or ceramic cartridges).

Large quantities of the desired antibodies can also be obtained bymultiplying mammalian cells in vivo. For this purpose, hybridoma cellsproducing the desired antibodies are injected into histocompatiblemammals to cause growth of antibody-producing tumors. Optionally, theanimals are primed with a hydrocarbon, especially mineral oils such aspristane (tetramethyl-pentadecane), prior to the injection. After one tothree weeks, the antibodies are isolated from the body fluids of thosemammals. For example, hybridoma cells obtained by fusion of suitablemyeloma cells with antibody-producing spleen cells from Balb/c mice, ortransfected cells derived from hybridoma cell line Sp2/0 that producethe desired antibodies are injected intraperitoneally into Balb/c miceoptionally pre-treated with pristane. After one to two weeks, asciticfluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example,Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110;Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold SpringHarbor, the disclosures of which are all incorporated herein byreference. Techniques for the preparation of recombinant antibodymolecules are described in the above references and also in, for exampleWO97/08320; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,508,717; Smith,1985, Science, Vol. 225, pp 1315-1317; Parmley and Smith, 1988, Gene 73,pp 305-318; De La Cruz et al., 1988, Journal of Biological Chemistry,263 pp 4318-4322; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,223,409;WO88/06630; WO92/15679; U.S. Pat. No. 5,780,279; U.S. Pat. No.5,571,698; U.S. Pat. No. 6,040,136; Davis et al., 1999, CancerMetastasis Rev., 18(4):421-5; Taylor, et al., Nucleic Acids Research 20(1992): 6287-6295; Tomizuka et al., Proc. Natl. Academy of Sciences USA97(2) (2000): 722-727. The contents of all these references areincorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies,preferentially by immunofluorescent staining of CD200-positive cells, byimmunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or adot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culturesupernatants or in the ascitic fluid may be concentrated, e.g. byprecipitation with ammonium sulfate, dialysis against hygroscopicmaterial such as polyethylene glycol, filtration through selectivemembranes, or the like. If necessary and/or desired, the antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinitychromatography with one or more surface polypeptides derived from aCD200-positive cell line, or with Protein-A or -G.

Another embodiment provides a process for the preparation of a bacterialcell line secreting antibodies directed against CD200 in a suitablemammal. For example a rabbit is immunized with pooled samples fromCD200-positive tissue or cells or CD200 polypeptide or fragmentsthereof. A phage display library produced from the immunized rabbit isconstructed and panned for the desired antibodies in accordance withmethods well known in the art (such as, for example, the methodsdisclosed in the various references incorporated herein by reference).

Hybridoma cells secreting the monoclonal antibodies are also disclosed.The preferred hybridoma cells are genetically stable, secrete monoclonalantibodies described herein of the desired specificity, and can beactivated from deep-frozen cultures by thawing and recloning.

In another embodiment, a process is provided for the preparation of ahybridoma cell line secreting monoclonal antibodies against CD200. Inthat process, a suitable mammal, for example a Balb/c mouse, isimmunized with one or more polypeptides or antigenic fragments of CD200or with one or more polypeptides or antigenic fragments derived from aCD200-positive cell, the CD200-positive cell itself, or an antigeniccarrier containing a purified polypeptide as described.Antibody-producing cells of the immunized mammal are grown briefly inculture or fused with cells of a suitable myeloma cell line. The hybridcells obtained in the fusion are cloned, and cell clones secreting thedesired antibodies are selected. For example, spleen cells of Balb/cmice immunized with a CD200-positive Chronic Lymphocytic Leukemia (CLL)cell line are fused with cells of the myeloma cell line PAI or themyeloma cell line Sp2/0-Ag 14. The obtained hybrid cells are thenscreened for secretion of the desired antibodies and positive hybridomacells are cloned.

Preferred is a process for the preparation of a hybridoma cell line,characterized in that Balb/c mice are immunized by injectingsubcutaneously and/or intraperitoneally between 10⁶ and 10⁷ cells of aCD200-positive cell line several times, e.g. four to six times, overseveral months, e.g. between two and four months. Spleen cells from theimmunized mice are taken two to four days after the last injection andfused with cells of the myeloma cell line PAI in the presence of afusion promoter, preferably polyethylene glycol. Preferably, the myelomacells are fused with a three- to twenty-fold excess of spleen cells fromthe immunized mice in a solution containing about 30% to about 50%polyethylene glycol of a molecular weight around 4000. After the fusion,the cells are expanded in suitable culture media as describedhereinbefore, supplemented with a selection medium, for example HATmedium, at regular intervals in order to prevent normal myeloma cellsfrom overgrowing the desired hybridoma cells.

The antibodies and fragments thereof can be “chimeric”. Chimericantibodies and antigen-binding fragments thereof comprise portions fromtwo or more different species (e.g., mouse and human). Chimericantibodies can be produced with mouse variable regions of desiredspecificity spliced into human constant domain gene segments (forexample, U.S. Pat. No. 4,816,567). In this manner, non-human antibodiescan be modified to make them more suitable for human clinicalapplication.

The monoclonal antibodies of the present disclosure include “humanized”forms of the non-human (i.e., mouse) antibodies. Humanized orCDR-grafted mAbs are particularly useful as therapeutic agents forhumans because they are not cleared from the circulation as rapidly asmouse antibodies and do not typically provoke an adverse immunereaction. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a non-human source. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Methods ofpreparing humanized antibodies are generally well known in the art. Forexample, humanization can be essentially performed following the methodof Winter and co-workers (Jones et al., Nature 321:522-525 (1986);Reichmann et al., Nature, 332:323-327 (1988); Verheoeyen et al.,Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Also seeStaelens et al. 2006 Mol Immunol 43: 1243-1257. In particularembodiments, humanized forms of non-human (e.g., mouse) antibodies arehuman antibodies (recipient antibody) in which hypervariable (CDR)region residues of the recipient antibody are replaced by hypervariableregion residues from a non-human species (donor antibody) such as amouse, rat, rabbit, or non-human primate having the desired specificity,affinity, and binding capacity. In some instances, framework regionresidues of the human immunoglobulin are also replaced by correspondingnon-human residues (so called “back mutations”). In addition, phagedisplay libraries can be used to vary amino acids at chosen positionswithin the antibody sequence. The properties of a humanized antibody arealso affected by the choice of the human framework. Furthermore,humanized and chimerized antibodies can be modified to comprise residuesthat are not found in the recipient antibody or in the donor antibody inorder to further improve antibody properties, such as, for example,affinity or effector function.

Fully human antibodies are also provided in the disclosure. The term“human antibody” includes antibodies having variable and constantregions (if present) derived from human germline immunoglobulinsequences. Human antibodies can include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). However, the term “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences (i.e., humanized antibodies). Fully human or human antibodiesmay be derived from transgenic mice carrying human antibody genes(carrying the variable (V), diversity (D), joining (J), and constant (C)exons) or from human cells. For example, it is now possible to producetransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production (see, e.g., Jakobovits et al.,PNAS; 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immuno., 7:33 (1993); and Duchosal et al.Nature 355:258 (1992). Transgenic mice strain can be engineered tocontain gene sequences from unrearranged human immunoglobulin genes. Thehuman sequences may code for both the heavy and light chains of humanantibodies and would function correctly in the mice, undergoingrearrangement to provide a wide antibody repertoire similar to that inhumans. The transgenic mice can be immunized with the target protein(e.g., CD200, fragments thereof, or cells expressing CD200) to create adiverse array of specific antibodies and their encoding RNA. Nucleicacids encoding the antibody chain components of such antibodies may thenbe cloned from the animal into a display vector. Typically, separatepopulations of nucleic acids encoding heavy and light chain sequencesare cloned, and the separate populations then recombined on insertioninto the vector, such that any given copy of the vector receives arandom combination of a heavy and a light chain. The vector is designedto express antibody chains so that they can be assembled and displayedon the outer surface of a display package containing the vector. Forexample, antibody chains can be expressed as fusion proteins with aphage coat protein from the outer surface of the phage. Thereafter,display packages can be screened for display of antibodies binding to atarget.

In addition, human antibodies can be derived from phage-displaylibraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks etal., J. Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech14:309 (1996)). Synthetic phage libraries can be created which userandomized combinations of synthetic human antibody V-regions. Byselection on antigen fully human antibodies can be made in which theV-regions are very human-like in nature. See U.S. Pat. Nos. 6,794,132,6,680,209, 4,634,666, and Ostberg et al. (1983), Hybridoma 2:361-367,the contents of which are incorporated by reference.

For the generation of human antibodies, also see Mendez et al. NatureGenetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med.188:483-495 (1998), the disclosures of which are hereby incorporated byreference. Human antibodies are further discussed and delineated in U.S.Pat. Nos. 5,939,598 and 6,673,986. Also see U.S. Pat. Nos. 6,114,598,6,075,181, and 6,162,963, all filed Jun. 5, 1995. Also see U.S. Pat. No.6,150,584, filed Oct. 2, 1996 and U.S. Pat. Nos. 6,713,610 and 6,657,103as well as U.S. patent application Ser. Nos. 10/421,011 (US 2003-0229905A1), 10/455,013 (US 2004-0010810 A1), 10/627,250 (US 2004-0093622 A1),10/656,623 (US 2006-0040363 A1), 10/658,521 (US 2005-0054055 A1),10/917,703 (US 2005-0076395 A1) and 10/978,297 (US 2005-0287630 A1). Seealso PCT/US93/06926 filed on Jul. 23, 1993, European Patent No., EP 0463 151 B1, grant published Jun. 12, 1996, International PatentApplication No., WO 94/02602, published Feb. 3, 1994, InternationalPatent Application No., WO 96/34096, published Oct. 31, 1996, and WO98/24893, published Jun. 11, 1998. The disclosures of each of theabove-cited patents, applications, and references are herebyincorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, and 5,814,318 each to Lonberg and Kay, U.S. Pat. No.5,591,669 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367,5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and Dunn,and GenPharm International. Also see U.S. Pat. Nos. 5,569,825,5,877,397, 6,300,129, 5,874,299, 6,255,458, and 7,041,871, thedisclosures of which are hereby incorporated by reference. See alsoEuropean Patent No. 0 546 073 B1, International Patent Application Nos.WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al. (1992 Nuc. Acids. Res., 20: 6287),Chen et al. (1993 Int. Immunol. 5: 647), Tuaillon et al. (1993 PNAS USA.90: 3720-4), Choi et al., (1993 Nature Genetics 4: 117), Lonberg et al.(1994 Nature 368: 856-859), Taylor et al. (1994 International Immunology6: 579-591), and Tuaillon et al. (1995 J. Immunol. 154: 6453-65),Fishwild et al. (1996 Nature Biotechnology 14: 845), and Tuaillon et al.(2000 Eur J. Immunol. 10: 2998-3005), the disclosures of which arehereby incorporated by reference in their entirety.

In certain embodiments, de-immunized anti-CD200 antibodies orantigen-binding fragments thereof are provided. De-immunized antibodiesor antigen-binding fragments thereof may be modified so as to render theantibody or antigen-binding fragment thereof non-immunogenic, or lessimmunogenic, to a given species. De-immunization can be achieved bymodifying the antibody or antigen-binding fragment thereof utilizing anyof a variety of techniques known to those skilled in the art (sec e.g.,PCT Publication Nos. WO 04/108158 and WO 00/34317). For example, anantibody or antigen-binding fragment thereof may be de-immunized byidentifying potential T cell epitopes and/or B cell epitopes within theamino acid sequence of the antibody or antigen-binding fragment thereofand removing one or more the potential T cell epitopes and/or B cellepitopes from the antibody or antigen-binding fragment thereof, forexample, using recombinant techniques. The modified antibody orantigen-binding fragment thereof may then optionally be produced andtested to identify antibodies or antigen-binding fragments thereof thathave retained one or more desired biological activities, such as, forexample, binding affinity, but have reduced immunogenicity. Methods foridentifying potential T cell epitopes and/or B cell epitopes may becarried out using techniques known in the art, such as, for example,computational methods (see e.g., PCT Publication No. WO 02/069232), invitro or in silico techniques, and biological assays or physical methods(such as, for example, determination of the binding of peptides to MHCmolecules, determination of the binding of peptide:MHC complexes to theT cell receptors from the species to receive the antibody orantigen-binding fragment thereof, testing of the protein or peptideparts thereof using transgenic animals with the MHC molecules of thespecies to receive the antibody or antigen-binding fragment thereof, ortesting with transgenic animals reconstituted with immune system cellsfrom the species to receive the antibody or antigen-binding fragmentthereof, etc.). In various embodiments, the de-immunized anti-CD200antibodies described herein include de-immunized antigen-bindingfragments, Fab, Fv scFv, Fab′ and F(ab′)₂, monoclonal antibodies, murineantibodies, engineered antibodies (such as, for example, chimeric,single chain, CDR-grafted, humanized, fully human antibodies, andartificially selected antibodies), synthetic antibodies andsemi-synthetic antibodies.

In a further embodiment, recombinant DNA comprising an insert coding fora heavy chain variable domain and/or for a light chain variable domainof antibodies directed to CD200 or a CD200-positive cell line areproduced. The term DNA includes coding single stranded DNAs, doublestranded DNAs consisting of said coding DNAs and of complementary DNAsthereto, or these complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or a lightchain variable domain of antibodies directed to CD200 or theCD200-positive cell line can be enzymatically or chemically synthesizedDNA having the authentic DNA sequence coding for a heavy chain variabledomain and/or for the light chain variable domain, or a mutant thereof.A mutant of the authentic DNA is a DNA encoding a heavy chain variabledomain and/or a light chain variable domain of the above-mentionedantibodies in which one or more amino acids are deleted, inserted, orexchanged with one or more other amino acids. Preferably saidmodification(s) are outside the CDRs of the heavy chain variable domainand/or of the light chain variable domain of the antibody inhumanization and expression optimization applications. The term mutantDNA also embraces silent mutants wherein one or more nucleotides arereplaced by other nucleotides with the new codons coding for the sameamino acid(s). The term mutant sequence also includes a degeneratesequence. Degenerate sequences are degenerate within the meaning of thegenetic code in that an unlimited number of nucleotides are replaced byother nucleotides without resulting in a change of the amino acidsequence originally encoded. Such degenerate sequences may be useful dueto their different restriction sites and/or frequency of particularcodons which are preferred by the specific host, particularly E. coli,to obtain an optimal expression of the heavy chain murine variabledomain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitromutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of chimeric antibodies, the recombinant DNA inserts codingfor heavy and light chain variable domains are fused with thecorresponding DNAs coding for heavy and light chain constant domains,then transferred into appropriate host cells, for example afterincorporation into hybrid vectors.

Recombinant DNAs including an insert coding for a heavy chain murinevariable domain of an antibody directed to CD200 or a CD200-positivecell line fused to a human constant domain IgG, for example γ1, γ2, γ3or γ4; in particular embodiments γ1 or γ4 may be used. Recombinant DNAsincluding an insert coding for a light chain murine variable domain ofan antibody directed to the cell line disclosed herein fused to a humanconstant domain κ or λ, preferably κ are also provided.

Another embodiment pertains to recombinant DNAs coding for a recombinantpolypeptide wherein the heavy chain variable domain and the light chainvariable domain are linked by way of a spacer group, optionallycomprising a signal sequence facilitating the processing of the antibodyin the host cell and/or a DNA sequence encoding a peptide facilitatingthe purification of the antibody and/or a cleavage site and/or a peptidespacer and/or an agent. The DNA coding for an agent is intended to be aDNA coding for the agent useful in diagnostic or therapeuticapplications. Thus, agent molecules which are toxins or enzymes,especially enzymes capable of catalyzing the activation of prodrugs, areparticularly indicated. The DNA encoding such an agent has the sequenceof a naturally occurring enzyme or toxin encoding DNA, or a mutantthereof, and can be prepared by methods well known in the art.

Accordingly, the monoclonal antibodies or antigen-binding fragments ofthe disclosure can be naked antibodies or antigen-binding fragments thatare not conjugated to other agents, for example, a therapeutic agent ordetectable label. Alternatively, the monoclonal antibody orantigen-binding fragment can be conjugated to an agent such as, forexample, a cytotoxic agent, a small molecule, a hormone, an enzyme, agrowth factor, a cytokine, a ribozyme, a peptidomimetic, a chemical, aprodrug, a nucleic acid molecule including coding sequences (such asantisense, RNAi, gene-targeting constructs, etc.), or a detectable label(e.g., an NMR or X-ray contrasting agent, fluorescent molecule, etc.).In certain embodiments, an anti-CD200 polypeptide or antigen-bindingfragment (e.g., Fab, Fv, single-chain scFv, Fab′ and F(ab′)₂) is linkedto a molecule that increases the half-life of the said polypeptide orantigen-binding fragment. Molecules that may be linked to saidanti-CD200 polypeptide or antigen-binding fragment include but are notlimited to serum proteins including albumin, polypeptides, otherproteins or protein domains, and PEG.

Several possible vector systems are available for the expression ofcloned heavy chain and light chain genes in mammalian cells. One classof vectors relies upon the integration of the desired gene sequencesinto the host cell genome. Cells which have stably integrated DNA can beselected by simultaneously introducing drug resistance genes such as Ecoli gpt (Mulligan, R. C. and Berg, P., Proc. Natl. Acad. Sci., USA, 78:2072 (1981)) or Tn5 neo (Southern, P. J. and Berg, P., J. Mol. Appl.Genet., 1: 327 (1982)). The selectable marker gene can be either linkedto the DNA gene sequences to be expressed, or introduced into the samecell by co-transfection (Wigler, M. et al., Cell, 16: 77 (1979)). Asecond class of vectors utilizes DNA elements which confer autonomouslyreplicating capabilities to an extrachromosomal plasmid. These vectorscan be derived from animal viruses, such as bovine papillomavirus(Sarver, N. et al., Proc. Natl. Acad. Sci., USA, 79: 7147 (1982)),polyoma virus (Deans, R. J. et al., Proc. Natl. Acad. Sci., USA, 81:1292 (1984)), or SV40 virus (Lusky, M. and Botchan, M., Nature, 293: 79(1981)).

Since an immunoglobulin cDNA is comprised only of sequences representingthe mature mRNA encoding an antibody protein, additional gene expressionelements regulating transcription of the gene and processing of the RNAare required for the synthesis of immunoglobulin mRNA. These elementsmay include splice signals, transcription promoters, including induciblepromoters enhancers, and termination signals. cDNA expression vectorsincorporating such elements include those described by Okayama, H. andBerg, P., Mol. Cell. Biol., 3: 280 (1983); Cepko, C. L. et al., Cell,37: 1053 (1984); and Kaufman, R. J., Proc. Natl. Acad. Sci., USA, 82:689 (1985).

In certain embodiments, an anti-CD200 antibody may be a blocking ornon-blocking antibody. As used herein, a blocking antibody is one thatblocks the interaction between CD200 and CD200R. A non-blocking antibodybinds and/or interacts with CD200 but does not block its interactionwith CD200R. Thus in certain embodiments, an anti-CD200 antibody iseither a blocking or non-blocking murine, chimeric, humanized human orde-immunized antibody.

II. CD200 Antagonists with Altered Effector Functions

CD200 antagonists may be altered to elicit increased or decreasedeffects relative to the original or parent antagonist. For example, anantagonist that binds CD200 may elicit secondary functions followingbinding to CD200 and, in some instances, inhibiting the CD200:CD200Rinteraction. For example, an antagonist may contain additional bindingsites for other ligands, including receptors or extracellular proteins.Binding to these other ligands may trigger other events, such as theattraction or recruitment of other cells and the activation of variousevents including cell death. Thus in certain aspects, the presentdisclosure relates to CD200 antagonists that elicit altered secondaryfunctions (or effector functions as referred to below). In certainembodiments, the CD200 antagonist with altered secondary or effectorfunction(s) exhibits increased, decreased, or no secondary or effectorfunction(s), and further may or may not block the CD200:CD200Rinteraction. In particular embodiments, the CD200 antagonist withaltered secondary or effector function(s) is an anti-CD200 antibody.

A) Effector Functions

The interaction of antibodies and antibody-antigen complexes with cellsof the immune system affects a variety of responses, referred to hereinas effector functions. Exemplary effector functions include Fc receptorbinding, phagocytosis, down-regulation of cell surface receptors (e.g. Bcell receptor; BCR), etc. Other effector functions include ADCC, wherebyantibodies bind Fc receptors on natural killer (NK) cells or macrophagesleading to cell death, and CDC, which is cell death induced viaactivation of the complement cascade (reviewed in Daeron, Annu. Rev.Immunol. 15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol.2:77-94 (1995); and Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492(1991)). Such effector functions generally require the Fc region to becombined with a binding domain (e.g. an antibody variable domain) andcan be assessed using various assays as herein disclosed, for example.

Several antibody effector functions, including ADCC, are mediated by Fcreceptors (FcRs), which bind the Fc region of an antibody. In ADCC, NKcells or macrophages bind to the Fc region of the antibody complex andpromote lysis of the target cell. The cross-linking of FcRs on NK cellstriggers perforin/granzyme-mediated cytotoxicity, whereas in macrophagesthis cross-linking promotes the release of mediators such as nitricoxide (NO), TNF-α, and reactive oxygen species. For CD200-positivetarget cells, an anti-CD200 antibody binds to the target cell and the Fcregion directs effector function to the target cell. The affinity of anantibody for a particular FcR, and hence the effector activity mediatedby the antibody, may be modulated by altering the amino acid Sequenceand/or post-translational modifications of the Fc and/or constant regionof the antibody.

FcRs are defined by their specificity for immunoglobulin isotypes; Fcreceptors for IgG antibodies are referred to as FcγR, for IgE as FcεR,for IgA as FcαR and so on. Three subclasses of FcγR have beenidentified: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Because eachFcγR subclass is encoded by two or three genes, and alternative RNAsplicing leads to multiple transcripts, a broad diversity in FcγRisoforms exists. The three genes encoding the FcγRI subclass (FcγRIA,FcγRIB and FcγRIC) are clustered in region 1q21.1 of the long arm ofchromosome 1; the genes encoding FcγRII isoforms (FcγRIIA, FcγRIIB andFcγRIIC) and the two genes encoding FcγRIII (FcγRIIIA and FcγRIIIB) areall clustered in region 1q22. These different FcR subtypes are expressedon different cell types (reviewed in Ravetch and Kinet, Arum. Rev.Immunol. 9:457-492 (1991)). For example, in humans, FcγRIIIB is foundonly on neutrophils, whereas FcγRIIIA is found on macrophages,monocytes, natural killer (NK) cells, and a subpopulation of T-cells.Notably, FcγRIIIA is the only FcR present on NK cells, one of the celltypes implicated in ADCC.

FcγRI, FcγRII and FcγRIII are immunoglobulin superfamily (IgSF)receptors; FcγRI has three IgSF domains in its extracellular domain,while FcγRII and FcγRIII have only two IgSF domains in theirextracellular domains. Another type of Fc receptor is the neonatal Fcreceptor (FcRn). FcRn is structurally similar to majorhistocompatibility complex (MHC) and consists of an α-chainnoncovalently bound to β2-microglobulin.

The binding site on human and murine antibodies for FcγR have beenpreviously mapped to the so-called “lower hinge region” consisting ofresidues 233-239 (EU index numbering as in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Woof et al. Molec.Immunol. 23:319-330 (1986); Duncan et al. Nature 332:563 (1988);Canfield and Morrison, J. Exp. Med. 173:1483-1491 (1991); Chappel etal., Proc. Natl. Acad. Sci. USA 88:9036-9040 (1991). Of residues233-239, P238 and S239 have been cited as possibly being involved inbinding.

Other previously cited areas possibly involved in binding to FcγR are:G316-K338 (human IgG) for human FcγRI (by sequence comparison only; nosubstitution mutants were evaluated) (Woof et al. Molec Immunol.23:319-330 (1986)); K274-R301 (human IgG1) for human FcγRIII (based onpeptides) (Sarmay et al. Molec. Immunol. 21:43-51 (1984)); Y407-R416(human IgG) for human FcγRIII (based on peptides) (Gergely et al.Biochem. Soc. Trans. 12:739-743 (1984)); as well as N297 and E318(murine IgG2b) for murine FcγRII (Lund et al., Molec. Immunol., 29:53-59(1992)).

Human effector cells are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function. Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils. Effector cells may be isolated from a native sourcethereof, e.g. from blood or PBMCs.

In CDC, the antibody-antigen complex binds complement, resulting in theactivation of the complement cascade and generation of the membraneattack complex. Activation of the classical complement pathway isinitiated by the binding of the first component of the complement system(C1q) to antibodies (of the appropriate subclass) which are bound totheir cognate antigen; thus the activation of the complement cascade isregulated in part by the binding affinity of the immunoglobulin to C1qprotein. C1q and two serine proteases, C1r and C1s, form the complex C1,the first component of the CDC pathway. C1q is a hexavalent moleculewith a molecular weight of approximately 460,000 and a structure inwhich six collagenous “stalks” are connected to six globular headregions. Burton and Woof, Advances in Immunol. 51:1-84 (1992). Toactivate the complement cascade, it is necessary for C1q to bind to atleast two molecules of IgG1, IgG2, or IgG3, but only one molecule ofIgM, attached to the antigenic target (Ward and Ghetie, TherapeuticImmunology 2:77-94 (1995) p. 80). To assess complement activation, a CDCassay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods202:163 (1996), may be performed.

It has been proposed that various residues of the IgG molecule areinvolved in binding to C1q including the Glu318, Lys320 and Lys322residues on the CH2 domain, amino acid residue 331 located on a turn inclose proximity to the same beta strand, the Lys235 and Gly237 residueslocated in the lower hinge region, and residues 231 to 238 located inthe N-terminal region of the CH2 domain (see e.g., Xu et al., J.Immunol. 150:152 A (Abstract) (1993) WO94/29351; Tao et al, J. Exp.Med., 178:661-667 (1993); Brekke et al., Eur. J. Immunol, 24:2542-47(1994); Burton et al; Nature, 288:338-344 (1980); Duncan and Winter,Nature 332:738-40 (1988); U.S. Pat. No. 5,648,260, and U.S. Pat. No.5,624,821). It has further been proposed that the ability of IgG to bindC1q and activate the complement cascade also depends on the presence,absence or modification of the carbohydrate moiety positioned betweenthe two CH2 domains (which is normally anchored at Asn297) (Ward andGhetie, Therapeutic Immunology 2:77-94 (1995). In certain embodiments,one or more of these residues may be modified, substituted, or removedor one or more amino acid residues may be inserted so as to enhance ordecrease CDC activity of the anti-CD200 antibodies provided herein.

B) Anti-CD200 Antibodies with Modulated Effector Function(s)

Effector functions involving the constant region of the target-specificantibody may be modulated by altering properties of the constant or Fcregion. Altered effector functions include, for example, a modulation inone or more of the following activities: ADCC, CDC, apoptosis, bindingto one or more Fc-receptors, and pro-inflammatory responses. Modulationrefers to an increase, decrease, or elimination of an activity comparedto the activity of a second antibody. In certain embodiments, the secondantibody is an antibody with effector function. The second antibody maybe an engineered antibody or a naturally occurring antibody and may bereferred to as a non-variant, native, or parent antibody. In particularembodiments, modulation includes situations in which an activity isabolished or completely absent. Further, in some instances, anon-variant antibody may exhibit effector function activity similar orequivalent to the activity of the chC2aB7-hG1 or the hB7V3V2-hG1antibodies disclosed herein. Likewise, a functional or non-variantconstant or Fc region may possess an effector function of a nativeconstant or Fc domain; in some instances, the constant or Fc region ofchC2aB7-hG1 or hB7V3V2-hG1 may represent the non-variant domains. Forpresent purposes, chC2aB7-hG1 and hB7V3V2-hG1 are the standards againstwhich the activities of other antibodies are compared, with hB7V3V2-hG1being the preferred standard.

A polypeptide variant with altered FcR binding affinity and/or ADCCactivity and/or altered CDC activity is a polypeptide which has eitherenhanced or diminished FcR binding activity and/or ADCC activity and/orCDC activity compared to the native or parent polypeptide or to apolypeptide comprising a native sequence Fc or constant region. Apolypeptide variant which displays increased binding to an FcR binds atleast one FcR with greater affinity than the parent polypeptide. Apolypeptide variant which displays decreased binding to an FcR binds atleast one FcR with lower affinity than a parent polypeptide. Suchvariants which display decreased binding to an FcR may possess little orno appreciable binding to an FcR, e.g., 0-20% binding to the FcRcompared to the level of binding of a native sequence immunoglobulinconstant or Fc region to the FcR. Similarly a polypeptide variant whichdisplays altered ADCC and/or CDC activity may exhibit either increasedor reduced ADCC and/or CDC activity compared to the native or parentpolypeptide. A polypeptide variant which displays reduced ADCC and/orCDC may exhibit reduced or no ADCC and/or CDC activity as shown hereinby example. In certain embodiments, the parent or native polypeptide andits variant are antibodies or antigen-binding fragments. In particularembodiments, the said antibody or antigen-binding fragment binds CD200and may or may not block the CD200:CD200R interaction.

A native sequence Fc or constant region comprises an amino acid sequenceidentical to the amino acid sequence of a Fc or constant chain regionfound in nature. A variant or altered Fc or constant region comprises anamino acid sequence which differs from that of a native sequence heavychain region by virtue of at least one amino acid modification,insertion, or deletion, for example. In certain embodiments, the variantor altered constant region has at least one amino acid substitution,insertion, and/or deletion, compared to a native sequence constantregion or to the constant region of a parent polypeptide, e.g. fromabout one to about one hundred amino acid substitutions, insertions,and/or deletions in a native sequence constant region or in the constantregion of the parent polypeptide. In some embodiments, the variant oraltered constant region herein will possess at least about 70% homology(similarity) or identity with a native sequence constant region and/orwith a constant region of a parent polypeptide, and in some instances atleast about 75% and in other instances at least about 80% homology oridentity therewith, and in other embodiments at least about 85%, 90% or95% homology or identity therewith. The variant or altered constantregion may also contain one or more amino acid deletions or insertions.Additionally, the variant constant region may contain one or more aminoacid substitutions, deletions, or insertions that results in alteredpost-translational modifications, including, for example, an alteredglycosylation pattern.

Variant anti-CD200 antibodies as presently disclosed may be encoded by anucleic acid sequence that encodes a polypeptide with one or more aminoacid insertions, deletions, or substitutions relative to the native orparent polypeptide sequence. Furthermore, variant antibodies may beencoded by nucleic acid sequences that hybridize under stringentconditions to a nucleic acid sequence encoding a variant anti-CD200antibody. A variety of conditions may be used to detect hybridization,and the stringency is determined primarily by the wash stage of thehybridization assay. Generally high temperatures and low saltconcentrations give high stringency, while low temperatures and highsalt concentrations give low stringency. Low stringency hybridization isachieved by washing in, for example, about 2.0×SSC at 50° C., and highstringency is achieved with about 0.2×SSC at 50° C.

Antibodies or antigen-binding fragments thereof with altered or noeffector functions may be generated by engineering or producingantibodies with variant constant, Fc, or heavy chain regions;recombinant DNA technology and/or cell culture and expression conditionsmay be used to produce antibodies with altered function and/or activity.For example, recombinant DNA technology may be used to engineer one ormore amino acid substitutions, deletions, or insertions in regions (suchas, for example, Fc or constant regions) that affect antibody functionincluding effector functions. Alternatively, changes inpost-translational modifications, such as, e.g. glycosylation patterns,may be achieved by manipulating the cell culture and expressionconditions by which the antibody is produced.

Accordingly, certain aspects and methods of the present disclosurerelate to anti-CD200 antibodies with altered effector functions thatcomprise one or more amino acid substitutions, insertions, and/ordeletions. In certain embodiments, such a variant anti-CD200 antibodyexhibits reduced or no effector function. In particular embodiments, avariant antibody comprises a G2/G4 construct in place of the G1 domain(see FIGS. 10, 11, 12, 13, and 15, for example).

In addition to swapping the G1 domain with a G2/G4 construct aspresented herein, anti-CD200 antibodies with reduced effector functionmay be produced by introducing other types of changes in the amino acidsequence of certain regions of the antibody. Such amino acid sequencechanges include but are not limited to the Ala-Ala mutation described byBluestone et al. (see WO 94/28027 and WO 98/47531; also see Xu et al.2000 Cell Immunol 200; 16-26). Thus in certain embodiments, anti-CD200antibodies with mutations within the constant region including theAla-Ala mutation may be used to reduce or abolish effector function.According to these embodiments, the constant region of an anti-CD200antibody comprises a mutation to an alanine at position 234 or amutation to an alanine at position 235. Additionally, the constantregion may contain a double mutation: a mutation to an alanine atposition 234 and a second mutation to an alanine at position 235. In oneembodiment, the anti-CD200 antibody comprises an IgG4 framework, whereinthe Ala-Ala mutation would describe a mutation(s) from phenylalanine toalanine at position 234 and/or a mutation from leucine to alanine atposition 235. In another embodiment, the anti-CD200 antibody comprisesan IgG1 framework, wherein the Ala-Ala mutation would describe amutation(s) from leucine to alanine at position 234 and/or a mutationfrom leucine to alanine at position 235. An anti-CD200 antibody mayalternatively or additionally carry other mutations, including the pointmutation K322A in the CH2 domain (Hezareh et al. 2001 J Virol. 75:12161-8). An antibody with said mutation(s) in the constant region mayfurthermore be a blocking or non-blocking antibody.

Changes within the hinge region also affect effector functions. Forexample, deletion of the hinge region may reduce affinity for Fcreceptors and may reduce complement activation (Klein et al. 1981 PNASUSA 78: 524-528). The present disclosure therefore also relates toantibodies with alterations in the hinge region.

In particular embodiments, anti-CD200 antibodies may be modified toeither enhance or inhibit complement dependent cytotoxicity (CDC).Modulated CDC activity may be achieved by introducing one or more aminoacid substitutions, insertions, or deletions in an Fc region of theantibody (see, e.g., U.S. Pat. No. 6,194,551). Alternatively oradditionally, cysteine residue(s) may be introduced in the Fc region,thereby allowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated may have improved or reducedinternalization capability and/or increased or decreasedcomplement-mediated cell killing. See Caron et al., J. Exp Med.176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992),WO99/51642, Duncan & Winter Nature 322: 738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351. Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch 53:2560-2565 (1993). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989).

Another potential means of modulating effector function of antibodiesincludes changes in glycosylation. This topic has been recently reviewedby Raju who summarized the proposed importance of the oligosaccharidesfound on human IgGs with their degree of effector function (Raju, T S.BioProcess International April 2003. 44-53). According to Wright andMorrison, the microheterogeneity of human IgG oligosaccharides canaffect biological functions such as CDC and ADCC, binding to various Fcreceptors, and binding to C1q protein (Wright A. & Morrison S L. TIBTECH1997, 15 26-32). It is well documented that glycosylation patterns ofantibodies can differ depending on the producing cell and the cellculture conditions (Raju, T S. BioProcess International April 2003.44-53). Such differences can lead to changes in both effector functionand pharmacokinetics (Israel et al. Immunology. 1996; 89(4):573-578;Newkirk et al. P. Clin. Exp. 1996; 106(2):259-64). Differences ineffector function may be related to the IgGs ability to bind to the Fcγreceptors (FcγRs) on the effector cells. Shields, et al., have shownthat IgG, with variants in amino acid sequence that have improvedbinding to FcγR, can exhibit up to 100% enhanced ADCC using humaneffector cells (Shields et al. J Biol Chem. 2001 276(9):6591-604). Whilethese variants include changes in amino acids not found at the bindinginterface, both the nature of the sugar component as well as itsstructural pattern may also contribute to the differences observed. Inaddition, the presence or absence of fucose in the oligosaccharidecomponent of an IgG can improve binding and ADCC (Shields et al. J BiolChem. 2002; 277(30):26733-40). An IgG that lacked a fucosylatedcarbohydrate linked to Asn²⁹⁷ exhibited normal receptor binding to theFcγ receptor. In contrast, binding to the FcγRIIA receptor was improved50% and accompanied by enhanced ADCC, especially at lower antibodyconcentrations.

Work by Shinkawa, et al., demonstrated that an antibody to the humanIL-5 receptor produced in a rat hybridoma showed more than 50% higherADCC when compared to the antibody produced in Chinese hamster ovarycells (CHO) (Shinkawa et al. J Biol Chem. 2003 278(5):3466-73).Monosaccharide composition and oligosaccharide profiling showed that therat hybridoma-produced IgG had a lower content of fucose than theCHO-produced protein. The authors concluded that the lack offucosylation of an IgG 1 has a critical role in enhancement of ADCCactivity.

A different approach was taken by Umana, et al., who changed theglycosylation pattern of chCE7, a chimeric IgG1 anti-neuroblastomaantibody (Umana et al. Nat Biotechnol. 1999 February; 17(2): 176-80).Using tetracycline, they regulated the activity of a glycosyltransferaseenzyme (GnnII) which bisects oligosaccharides that have been implicatedin ADCC activity. The ADCC activity of the parent antibody was barelyabove background level. Measurement of ADCC activity of the chCE7produced at different tetracycline levels showed an optimal range ofGnTIH expression for maximal chCE7 in vitro ADCC activity. This activitycorrelated with the level of constant region-associated, bisectedcomplex oligosaccharide. Newly optimized variants exhibited substantialADCC activity. Similarly, Wright and Morrison produced antibodies in aCHO cell line deficient in glycosylation (1994 J Exp Med 180: 1087-1096)and showed that antibodies produced in this cell line were incapable ofcomplement-mediated cytolysis. Thus as known alterations that affecteffector function include modifications in the glycosylation pattern ora change in the number of glycosylated residues, the present disclosurerelates to a CD200 antibody wherein glycosylation is altered to eitherenhance or decrease effector function(s) including ADCC and CDC. Alteredglycosylation includes a decrease or increase in the number ofglycosylated residues as well as a change in the pattern or location ofglycosylated residues.

Still other approaches exist for the altering effector function ofantibodies. For example, antibody-producing cells can be hypermutagenic,thereby generating antibodies with randomly altered nucleotide andpolypeptide residues throughout an entire antibody molecule (see WO2005/011735). Hypermutagenic host cells include cells deficient in DNAmismatch repair. Antibodies produced in this manner may be lessantigenic and/or have beneficial pharmacokinetic properties.Additionally, such antibodies may be selected for properties such asenhanced or decreased effector function(s).

It is further understood that effector function may vary according tothe binding affinity of the antibody. For example, antibodies with highaffinity may be more efficient in activating the complement systemcompared to antibodies with relatively lower affinity (Marzocchi-Machadoet al. 1999 Immunol Invest 28: 89-101). Accordingly, an antibody may bealtered such that the binding affinity for its antigen is reduced (e.g.,by changing the variable regions of the antibody by methods such assubstitution, addition, or deletion of one or more amino acid residues).An anti-CD200 antibody with reduced binding affinity may exhibit reducedeffector functions, including, for example, reduced ADCC and/or CDC.

III. Methods of Depleting or Eliminating Cells Overexpressing CD200

In accordance with the present disclosure, methods are provided fordepleting cells that express CD200 in a subject by administering to thesubject a therapy comprising a CD200 antagonist. As mentioned above,CD200 is expressed on certain immune cells; and as demonstrated in thepresent disclosure, CD200 is also expressed on certain malignant cells.The disparate expression of CD200 provides an avenue by which to targetcancer cells (i.e., CD200-positive cells) for therapy. Likewise,CD200-positive immune cells may be targeted for depletion in methods oftreating autoimmune disorders.

CD200, through its interaction with CD200R on myeloid cells, modulatesimmunosuppression by delivering an inhibitory signal for myeloidactivity and/or migration. CD200-knockout mice, for example, demonstratea more active immune response following immunogenic stimuli (Hoek et al.Science 2000), and CD200-expressing cells elicit immunosuppression byinducing a shift in the cytokine profile of stimulated immune cells (seedata shown herein). Specifically, CD200-positive cells are capable ofinducing a shift from Th1 to Th2 cytokine production in mixed cellpopulation assays. While CD200-positive cells are capable of suppressingthe immune response, CD200-positive cancer cells, accordingly, may becapable of escaping immune cell attack. However expression of CD200 onthe membrane of cancer cells as well as immune cells can be exploited totarget these cells in therapy. For example, an anti-CD200 antagonist canspecifically target CD200-positive cells and disrupt the CD200:CD200Rinteraction, thereby inhibiting immune suppression, as well as targetCD200-positive cells to immune effector cells. The embodiments of thisdisclosure, therefore, relate to methods of targeting CD200-positivecells for depletion comprising an antagonist that binds to CD200 and, insome instances, disrupts the CD200:CD200R interaction.

In certain embodiments, the present disclosure relates to methods ofenhancing the immune response. Such methods include administering atherapy comprising a CD200 antagonist, and in particular embodiments theantagonist is an anti-CD200 antibody or antigen-binding fragment as setforth herein. While not wishing to be bound by any particularmechanism(s), a blocking anti-CD200 antibody, antigen-binding fragment,polypeptide, or other antagonist may eliminate CD200-positive cells byblocking immune suppression, thereby allowing immune cells to attack andeliminate CD200-positive cells. Alternatively or in combination with theaforementioned mechanism, an anti-CD200 antibody (either blocking ornon-blocking) or other antagonist may recruit effector cells or otherligands (e.g., complement component) to the CD200-positive cell to whichthe antibody or antagonist is bound and target the CD200-positive cellfor effector-mediated cell death.

In one aspect, the present disclosure relates to methods of modulatingADCC and/or CDC of CD200-positive target cells by administering amurine, chimeric, humanized, or human anti-CD200 antibody to a subjectin need thereof. The disclosure relates to variant anti-CD200 antibodiesthat elicit increased ADCC and/or CDC and to variant anti-CD200antibodies that exhibit reduced or no ADCC and/or CDC activity.

In one embodiment, the variant anti-CD200 antibody comprises a variantor altered Fc or constant region, wherein the variant Fc or constantregion exhibits increased effector function. Such said variant regionmay contain one or more amino acid substitutions, insertions, ordeletions. Alternatively or additionally, the variant or altered Fc orconstant region may comprise altered post-translational modifications,including, for example, an altered glycosylation pattern. An alteredglycosylation pattern includes an increase or decrease in the number ofglycosydic bonds and/or a modification in the location (i.e., amino acidresidue number) of one or more glycosydic bonds.

In another embodiment, the disclosure relates to methods of depleting oreliminating CD200-positive cells comprising variant anti-CD200antibodies that exhibit reduced or no ADCC and/or CDC activity. In oneembodiment, the variant anti-CD200 antibody comprises a variant oraltered Fc or constant region, wherein the variant Fc or constant regionexhibits decreased or no effector function. Such said variant or alteredFc or constant region may contain one or more amino acid substitutions,insertions, or deletions. Alternatively or additionally, the variant Fcor constant region may comprise altered post-translationalmodifications, including but not limited to an altered glycosylationpattern. Examples of altered glycosylation patterns are described above.

In a further embodiment, a murine, chimeric, humanized, human orde-immunized anti-CD200 antibody administered to a patient is anon-blocking antibody. The non-blocking anti-CD200 antibody may be avariant antibody as described above and may consequently exhibitmodulated effector function(s). For example, a variant anti-CD200antibody may not block the CD200:CD200R interaction and may alsocomprise a variant constant region that elicits increased effectorfunction, such as, e.g., increased ADCC.

A) Methods of Treating Patients with Autoimmune Disorders

In certain aspects, the disclosure relates to treating patients withautoimmune disorders with a therapy comprising a CD200 antagonist. Incertain embodiments, the antagonist is an anti-CD200 antibody orantigen-binding fragment thereof. In other embodiments, the anti-CD200antibody or fragment thereof is a variant anti-CD200 antibody thatexhibits modulated effector activity. For example, the variant antibodymay comprise a variant or altered constant region capable of elicitingincreased or enhanced effector function, such as, for example, ADCC.Additionally, the said antibody may be a non-blocking antibody and maybe a murine, chimeric, humanized, human or de-immunized anti-CD200antibody. Thus, methods of treating patients with autoimmune disordersmay comprise any of the CD200 antagonists and antibodies as set forth inthe present disclosure.

In certain embodiments, anti-CD200 antibodies or CD200 antagonists maybe used for depleting any type of cell that expresses CD200 on itssurface, including for example, immune cells such as T-cells, B-cells,and dendritic cells. In one embodiment, anti-CD200 antibodies may beuseful for targeted destruction of immune cells involved in an unwantedimmune response, such as, for example, immune responses associated withan autoimmune disorder, transplants, allergies, or inflammatorydisorders. Exemplary autoimmune diseases and disorders that may betreated with the anti-CD200 antibodies provided herein include, forexample, inflammatory responses such as inflammatory skin diseasesincluding psoriasis and dermatitis (e.g. atopic dermatitis);dermatomyositis; systemic scleroderma and sclerosis; responsesassociated with inflammatory bowel disease (such as Crohn's disease andulcerative colitis); respiratory distress syndrome (including adultrespiratory distress syndrome; ARDS); dermatitis; meningitis;encephalitis; uveitis; colitis; glomerulonephritis; allergic conditionssuch as eczema and asthma and other conditions involving infiltration ofT cells and chronic inflammatory responses; atherosclerosis; leukocyteadhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus(SLE); diabetes mellitus (e.g. Type I diabetes mellitus or insulindependent diabetes mellitis); multiple sclerosis; Reynaud's syndrome;autoimmune thyroiditis; allergic encephalomyelitis; Sjogren's syndrome;juvenile onset diabetes; and immune responses associated with acute anddelayed hypersensitivity mediated by cytokines and T-lymphocytestypically found in tuberculosis, sarcoidosis, polymyositis,granulomatosis and vasculitis; pernicious anemia (Addison's disease);diseases involving leukocyte diapedesis; central nervous system (CNS)inflammatory disorder; multiple organ injury syndrome; hemolytic anemia(including, but not limited to cryoglobinemia or Coombs positiveanemia); myasthenia gravis; antigen-antibody complex mediated diseases;anti-glomerular basement membrane disease; antiphospholipid syndrome;allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome;pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; Reiter'sdisease; stiff-man syndrome; Bechet disease; giant cell arteritis;immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia andautoimmune hemolytic diseases, Hashimoto's thyroiditis, etc.

In accordance with the methods and compositions described herein, thedisclosure also relates to methods of treating a transplant or allograftpatient. An anti-CD200 antibody or other CD200 antagonist of the presentdisclosure may be administered to a patient prior to a transplant orallograft procedure or after the procedure in order to decrease oreliminate CD200-positive immune cells that could reduce the patient'sacceptance of the transplanted organ or tissue. In a particularembodiment, an anti-CD200 antibody with increased effector function isgiven to a transplant patient. In addition, an anti-CD200 antibody is anon-blocking antibody.

Therapies comprising CD200 antagonists or antibodies may be administeredto patients in combination therapies. Accordingly, targeted killing ofcertain populations of immune cells for treating or preventingautoimmune disorders, enhancing or extending transplant survival,treating or preventing allergies, or treating or preventing inflammatorydisorders, may be administered as part of a combination therapy. Forexample, a patient receiving a first therapy comprising a CD200antagonist (e.g., an anti-CD200 antibody described herein) may also begiven a second therapy. The CD200 antagonist may be given simultaneouslywith the second therapy. Alternatively, the CD200 antagonist may begiven prior to or following the second therapy. Second therapies includebut are not limited to anti-inflammatory agents, immunosuppressiveagents, and/or anti-infective agents.

Combination therapies of the present disclosure include, for example, aCD200 antagonist as described herein administered concurrently orsequentially in series with steroids, anti-malarials, aspirin,non-steroidal anti-inflammatory drugs, immunosuppressants, or cytotoxicdrugs. Included are corticosteroids (e.g. prednisone, dexamethasone, andpredisolone), methotrexatem, methylprednisolone, macrolideimmunosuppressants (e.g. sirolimus and tacrolimus), mitotic inhibitors(e.g. azathioprine, cyclophosphamide, and methotrexate), fungalmetabolites that inhibit the activity of T lymphocytes (e.g.cyclosporine), mycophenolate mofetil, glatiramer acetate, and cytotoxicand DNA-damaging agents (e.g. chlorambucil). For autoimmune disordersand allograft or transplant patients, anti-CD200 therapy may be combinedwith antibody treatments including daclizumab, a genetically engineeredhuman IgG1 monoclonal antibody that binds specifically to the α-chain ofthe interleukin-2 receptor, as well as various other antibodiestargeting immune cells or other cells. Such combination therapies may beuseful in the treatment of type 1 diabetes, rheumatoid arthritis, lupus,and idiopathic thrombocytopenic purpura, and other autoimmuneindications. The disclosure also relates to therapies for autoimmunedisorders and for transplant patients comprising a CD200 antagonist(such as, for example, the antibodies and variants thereof described inthe present disclosure) conjugated to one or more agent.

B) Methods of Treating Patients with Cancer

In one aspect, the disclosure provides a method of treating cancer inwhich an agent that disrupts or inhibits the interaction of CD200 withits receptor is administered to a subject. Disruption of theCD200:CD200R interaction subsequently reverses or inhibits immunesuppression, thus enhancing the immune response. Possible agents for thedisruption of the CD200:CD200R interaction include, for example, smallmolecules, chemicals, polypeptides, inorganic molecules, andorganometallic compounds. The CD200:CD200R interaction may also beinhibited by reducing the expression of either the membrane protein orits receptor via antisense, RNAi, or gene therapy. Additionally, apolypeptide specific for CD200 or CD200R, such as an anti-CD200- oranti-CD200R-specific antibody or fragments thereof, may inhibit theimmunosuppressive effects of the CD200:CD200R interaction.

Cancer cells that may be treated by a CD200 antagonist include anycancer cells that exhibit CD200 expression or CD200 up-regulation.Cancers for which anti-CD200 therapy may be used include, for example,ovarian, melanoma, myeloma, neuroblastoma, renal, breast, prostate,hematological malignancies (e.g., lymphomas and leukemias), and plasmacell cancer. Also included are any cancer cells derived from neuralcrest cells. In some embodiments, the CD200 antagonist is an anti-CD200antibody. Such antibodies used as anti-cancer therapeutics are capableof interfering with the interaction of CD200 and its receptors. Thisinterference can block the immune-suppressing effect of CD200. Byimproving the immune response in this manner, such antibodies canpromote the eradication of cancer cells. Anti-CD200 antibodies may alsotarget cancer cells for effector-mediated cell death.

In one embodiment, a variant anti-CD200 antibody that exhibits modulatedADCC and/or CDC activity may be administered to a subject withCD200-positive cancer cells. For example, a variant anti-CD200 antibodyused in cancer therapy may exhibit enhanced effector activity comparedto the parent or native antibody. In another embodiment, the variantanti-CD200 antibody exhibits reduced effector function, includingreduced ADCC, relative to the native antibody. The said antibody may bea murine, chimeric, humanized, human or de-immunized antibody. Cancersfor which the variant anti-CD200 antibody may be used in treatmentinclude but are not limited to neural crest cell cancers. Also includedare plasma cell cancer, ovarian cancer, skin cancer, lung cancer, renalcancer, breast cancer, prostate cancer, neuroblastoma, lymphoma,myeloma, and leukemia.

The present antibodies can be administered as a therapeutic to cancerpatients, especially, but not limited to, patients with CLL, plasma cellcancer, ovarian cancer, skin cancer, lung cancer, renal cancer, breastcancer, prostate cancer, neuroblastoma, lymphoma, myeloma, leukemia, andany cancer derived from neural crest cells. In a particularly usefulembodiment, a cancer therapy in accordance with this disclosurecomprises (1) administering an anti-CD200 antibody or antagonist thatinterferes with the interaction between CD200 and its receptor to blockimmune suppression, thereby promoting eradication of the cancer cells;and/or (2) administering a fusion molecule that includes a CD-200targeting portion to directly kill cancer cells. Alternatively, theantibody directly kills the cancer cells through complement-mediated orantibody-dependent cellular cytotoxicity. Since CD200 is also expressedon normal cells such as endothelial cells, albeit at lower levels thanon cancer cells, it could also be advantageous to administer ananti-CD200 antibody with a constant region modified to reduce oreliminate ADCC or CDC to limit damage to normal cells. For example, ifCD200 expression is upregulated on some activated normal cells (e.g.,activated T cells), rendering such cells vulnerable to killing by ananti-CD200 antibody with effector function, it may therefore also beadvantageous to use an anti-CD200 antibody lacking effector function toavoid depletion of these cells which aid in destroying cancer cells.

In a particular embodiment, effector function of anti-CD200 antibodiesis eliminated by swapping the IgG1 constant domain for an IgG2/4 fusiondomain. Other ways of eliminating effector function can be envisionedsuch as, e.g., mutation of the sites known to interact with FcR orinsertion of a peptide in the hinge region, thereby eliminating criticalsites required for FcR interaction. Variant anti-CD200 antibodies withreduced or no effector function also include variants as describedpreviously herein.

The aforementioned agents for the inhibition or prevention of theCD200:CD200R interaction may be used in combination with other therapiesor with other agents. Other agents include but are not limited topolypeptides, small molecules, chemicals, metals, organometalliccompounds, inorganic compounds, nucleic acid molecules,oligonucleotides, aptamers, spiegelmers, antisense nucleic acids, lockednucleic acid (LNA) inhibitors, peptide nucleic acid (PNA) inhibitors,immunomodulatory agents, antigen-binding fragments, prodrugs, andpeptidomimetic compounds.

In certain aspects, the present disclosure relates to combinationtreatments comprising a CD200 antagonist including the antibodiesdescribed herein and immunomodulatory compounds, vaccines orchemotherapy. Illustrative examples of suitable immunomodulatory agentsthat may be used in such combination therapies include agents that blocknegative regulation of T cells or antigen presenting cells (e.g.,anti-CTLA4 antibodies, anti-PD-L1 antibodies, anti-PDL-2 antibodies,anti-PD-1 antibodies and the like) or agents that enhance positiveco-stimulation of T cells (e.g., anti-CD40 antibodies or anti 4-1BBantibodies) or agents that increase NK cell number or T-cell activity(e.g., anti-CD200 antibodies alone or in combination with inhibitorssuch as IMiDs, thalidomide, or thalidomide analogs). Furthermore,immunomodulatory therapy could include cancer vaccines such as dendriticcells loaded with tumor cells, proteins, peptides, RNA, or DNA derivedfrom such cells, patient derived heat-shocked proteins (hsp's) orgeneral adjuvants stimulating the immune system at various levels suchas CpG, Luivac, Biostim, Ribominyl, Imudon, Bronchovaxom or any othercompound or other adjuvant activating receptors of the innate immunesystem (e.g., toll like receptor agonist, anti-CTLA-4 antibodies, etc.).Also, immunomodulatory therapy could include treatment with cytokinessuch as IL-2, GM-CSF and IFN-gamma.

In additional embodiments, elimination of existing regulatory T cellswith reagents such as anti-CD25, fludarabine, or cyclophosphamide isachieved before starting anti-CD200 treatment. Also, therapeuticefficacy of myeloablative therapies followed by bone marrowtransplantation or adoptive transfer of T cells reactive with CLL cellsis enhanced by anti-CD200 therapy. In yet other embodiments, efficacy ofanti-CD200 treatment is improved by blocking immunosuppressivemechanisms with agents such as anti-PDL1 and/or 2 antibodies, anti-IL-10antibodies, anti-IL-6 antibodies, and the like. Furthermore, it could beadvantageous to eliminate plasmacytoid dendritic cells, shown to beimmunosuppressive in the cancer environment. In these embodiments inwhich delivery of an anti-CD200 antibody is intended to augment animmune response by blocking immune suppression, for example, a variantanti-CD200 antibody lacking effector function may also be used.

In particularly useful embodiments, the therapy that enhances immuneresponse is the administration of a polypeptide that binds to CD200,alone or in combination with one of the previously mentionedimmunomodulatory therapies. Accordingly, a CD200 antagonist (includingan anti-CD200 antibody as described herein) may be used in combinationwith a monoclonal antibody (e.g., rituximab, trastuzumab, alemtuzumab,cetuximab, or bevacizumab), including a conjugated monoclonal antibody(e.g., gemtuzumab ozogamicin, ibritumomab tiuxetan, or tositumomab).

Furthermore, combination of anti-CD200 therapy with chemotherapeuticscould be particularly useful to reduce overall tumor burden, to limitangiogenesis, to enhance tumor accessibility, to enhance susceptibilityto ADCC, to result in increased immune function by providing more tumorantigen, or to increase the expression of the T cell attractant LIGHT.When anti-CD200 therapy is administered to a subject in combination withanother conventional anti-neoplastic agent, either concomitantly orsequentially, anti-CD200 therapy may be shown to enhance the therapeuticeffect of either agent alone. Pharmaceutical compounds that may be usedfor combinatory anti-tumor therapy include, merely to illustrate:aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,bicalutamide, bleomycin, buserelin, busulfan, camptothecin,capecitabine, carboplatin, carmustine, chlorambucil, cisplatin,cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,estramustine, etoposide, exemestane, filgrastim, fludarabine,fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine,genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib,interferon, irinotecan, letrozole, leucovorin, leuprolide, levamisole,lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone,nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel,pamidronate, pentostatin, plicamycin, porfimer, procarbazine,raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide,teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride,topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine,and vinorelbine.

These chemotherapeutic anti-tumor compounds may be categorized by theirmechanism of action into groups, including, for example, the followingclasses of agents: anti-metabolites/anti-cancer agents, such aspyrimidine analogs (5-fluorouracil, floxuridine, capecitabine,gemcitabine and cytarabine) and purine analogs, folate antagonists andrelated inhibitors (mercaptopurine, thioguanine, pentostatin and2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitoticagents including natural products such as vinca alkaloids (vinblastine,vincristine, and vinorelbine), microtubule disruptors such as taxane(paclitaxel, docetaxel), vincristine, vinblastine, nocodazole,epothilones and navelbine, epidipodophyllotoxins (etoposide,teniposide), DNA damaging agents (actinomycin, amsacrine,anthracyclines, bleomycin, busulfan, camptothecin, carboplatin,chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin,daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin,iphosphamide, melphalan, mechlorethamine, mitomycin, mitoxantrone,nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide,triethylenethiophosphoramide and etoposide (VP16)); antibiotics such asdactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin),idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin(mithramycin) and mitomycin; enzymes (L-asparaginase which systemicallymetabolizes L-asparagine and deprives cells which do not have thecapacity to synthesize their own asparagine); antiplatelet agents;antiproliferative/antimitotic alkylating agents such as nitrogenmustards (mechlorethamine, cyclophosphamide and analogs, melphalan,chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine andthiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU)and analogs, streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);immunomodulatory agents (thalidomide and analogs thereof such aslenalidomide (Revlimid, CC-5013) and CC-4047 (Actimid)),cyclophosphamide; anti-angiogenic compounds (TNP-470, genistein) andgrowth factor inhibitors (vascular endothelial growth factor (VEGF)inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensinreceptor blocker; nitric oxide donors; anti-sense oligonucleotides;antibodies (trastuzumab); cell cycle inhibitors and differentiationinducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors(doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin,dactinomycin, eniposide, epirubicin, etoposide, idarubicin andmitoxantrone, topotecan, irinotecan), corticosteroids (cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisone, andprenisolone); growth factor signal transduction kinase inhibitors;mitochondrial dysfunction inducers and caspase activators; and chromatindisruptors.

In certain embodiments, pharmaceutical compounds that may be used forcombinatory anti-angiogenesis therapy include: (1) inhibitors of releaseof “angiogenic molecules,” such as bFGF (basic fibroblast growthfactor); (2) neutralizers of angiogenic molecules, such as anti-βbFGFantibodies; and (3) inhibitors of endothelial cell response toangiogenic stimuli, including collagenase inhibitor, basement membraneturnover inhibitors, angiostatic steroids, fungal-derived angiogenesisinhibitors, platelet factor 4, thrombospondin, arthritis drugs such asD-penicillamine and gold thiomalate, vitamin D₃ analogs,alpha-interferon, and the like. For additional proposed inhibitors ofangiogenesis, see Blood et al., Biochim. Biophys. Acta, 1032:89-118(1990), Moses et al., Science, 248:1408-1410 (1990), Ingber et al., Lab.Invest., 59:44-51 (1988), and U.S. Pat. Nos. 5,092,885, 5,112,946,5,192,744, 5,202,352, and 6,573,256. In addition, there are a widevariety of compounds that can be used to inhibit angiogenesis, forexample, peptides or agents that block the VEGF-mediated angiogenesispathway, endostatin protein or derivatives, lysine binding fragments ofangiostatin, melanin or melanin-promoting compounds, plasminogenfragments (e.g., Kringles 1-3 of plasminogen), troponin subunits,antagonists of vitronectin α_(v)β₃, peptides derived from Saposin B,antibiotics or analogs (e.g., tetracycline, or neomycin),dienogest-containing compositions, compounds comprising a MetAP-2inhibitory core coupled to a peptide, the compound EM-138, chalcone andits analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos.6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810,6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103,6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845.

Depending on the nature of the combinatory therapy, administration ofthe anti-CD200 antibody may be continued while the other therapy isbeing administered and/or thereafter. Administration of the antibody maybe made in a single dose, or in multiple doses. In some instances,administration of the anti-CD200 antibody is commenced at least severaldays prior to the conventional therapy, while in other instances,administration is begun either immediately before or at the time of theadministration of the conventional therapy. In some cases, theanti-CD200 antibody will be administered after other therapies, or itcould be administered alternating with other therapies.

The present antibodies can be utilized to directly kill or ablatecancerous cells in vivo. Direct killing involves administering theantibodies (which are optionally fused to a cytotoxic drug) to a subjectrequiring such treatment. Since the antibodies recognize CD200 on cancercells, any such cells to which the antibodies bind are destroyed. Wherethe antibodies are used alone to kill or ablate cancer cells, suchkilling or ablation can be effected by initiating endogenous host immunefunctions, such as CDC and/or ADCC. Assays for determining whether anantibody kills cells in this manner are within the purview of thoseskilled in the art.

Accordingly in one embodiment, the antibodies of the present disclosuremay be used to deliver a variety of cytotoxic compounds. Any cytotoxiccompound can be fused to the present antibodies. The fusion can beachieved chemically or genetically (e.g., via expression as a single,fused molecule). The cytotoxic compound can be a biological, such as apolypeptide, or a small molecule. As those skilled in the art willappreciate, for small molecules, chemical fusion is used, while forbiological compounds, either chemical or genetic fusion can be employed.

Non-limiting examples of cytotoxic compounds include therapeutic drugs,a compound emitting radiation, molecules of plants, fungal, or bacterialorigin, biological proteins, and mixtures thereof. The cytotoxic drugscan be intracellularly acting cytotoxic drugs, such as short-rangeradiation emitters, including, for example, short-range, high-energyα-emitters. Enzymatically active toxins and fragments thereof areexemplified by diphtheria toxin A fragment, nonbinding active fragmentsof diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin Achain, abrin A chain, modeccin A chain, alpha-sacrin, certain Aleuritesfordii proteins, certain Dianthin proteins, Phytolacca americanaproteins (PAP, PAPII and PAP-S), Morodica charantia inhibitor, curcin,crotin, Saponaria officinalis inhibitor, gelonin, mitogillin,restrictocin, phenomycin, and enomycin, for example. Procedures forpreparing enzymatically active polypeptides of the immunotoxins aredescribed in WO84/03508 and WO85/03508, which are hereby incorporated byreference. Certain cytotoxic moieties are derived from adriamycin,chlorambucil, daunomycin, methotrexate, neocarzinostatin, and platinum,for example.

Procedures for conjugating the antibodies with the cytotoxic agents havebeen previously described and are within the purview of one skilled inthe art.

Alternatively, the antibody can be coupled to high energy radiationemitters, for example, a radioisotope, such as ¹³¹I, a γ-emitter, which,when localized at the tumor site, results in a killing of several celldiameters. See, e.g., S. E. Order, “Analysis, Results, and FutureProspective of the Therapeutic Use of Radiolabeled Antibody in CancerTherapy”, Monoclonal Antibodies for Cancer Detection and Therapy, R. W.Baldwin et al. (eds.), pp 303-316 (Academic Press 1985), which is herebyincorporated by reference. Other suitable radioisotopes includeα-emitters, such as ²¹²Bi, ²¹³Bi, and ²¹¹At, and β-emitters, such as¹⁸⁶Re and ⁹⁰Y.

In some embodiments, present CD200 binding antibodies provide thebenefit of blocking immune suppression in CLL by targeting the leukemiccells directly through CD200. Specifically, stimulating the immunesystem can allow the eradication of CLL cells from the spleen and lymphnodes. Applicants are unaware of any successful eradication of CLL cellsfrom these microenvironments having been achieved with agents thatsimply target B cells (such as alemtuzumab). In contrast, CLL reactive Tcells can have better access to these organs than antibodies. In otherembodiments, direct cell killing is achieved by tagging the CLL cellswith anti-CD200 Abs.

According to the compositions and methods of the present disclosure, inparticularly useful embodiments, the combination of direct cell killingand driving the immune response towards a Th1 profile provides aparticularly powerful approach to cancer treatment. Thus, in oneembodiment, a cancer treatment is provided wherein an antibody orantibody fragment, which binds to CD200 and both a) blocks theinteraction between CD200 and its receptor and b) directly kills thecancer cells expressing CD200, is administered to a cancer patient. Themechanism by which the cancer cells are killed can include, but are notlimited to ADCC and/or CDC; fusion with a toxin; fusion with aradiolabel; fusion with a biological agent involved in cell killing,such as granzyme B or performin; fusion with a cytotoxic virus; fusionwith a cytokine such as TNF-α or IFN-α. In an alternative embodiment, acancer treatment involves administering an antibody that both a) blocksthe interaction between CD200 and its receptor and b) enhances cytotoxicT cell or NK cell activity against the tumor. Such enhancement of thecytotoxic T cell or NK cell activity may, for example, be combined byfusing the antibody with cytokines such as e.g. IL-2, IL-12, IL-18,IL-13, and IL-5. In addition, such enhancement may be achieved byadministration of an anti-CD200 antibody in combination with inhibitorssuch as IMiDs, thalidomide, or thalidomide analogs.

In yet another embodiment, the cancer treatment involves administeringan antibody that both (1) blocks the interaction between CD200 and itsreceptor and (2) attracts T cells to the tumor cells. T cell attractioncan be achieved by fusing the Ab with chemokines such as MIG, IP-10,I-TAC, CCL21, CCL5 or LIGHT. Also, treatment with chemotherapeutics canresult in the desired upregulation of LIGHT. The combined action ofblocking immune suppression and killing directly through antibodytargeting of the tumor cells is a unique approach that providesincreased efficacy.

Anti-CD200 antibodies in accordance with the present disclosure can alsobe used as a diagnostic tool. Biopsies or cancer cell tissue samples maybe tested for CD200 expression prior to treatment in order to predictthe efficacy of anti-CD200 therapy, alone or in combination with otheragents or methods (such as chemotherapeutic agents, radiation therapy,immunomodulatory therapy, etc.). For example, using blood obtained frompatients with hematopoietic cancers, expression of CD200 can beevaluated on cancer cells by FACS analysis using anti-CD200 antibodiesin combination with the appropriate cancer cell markers such as, e.g.,CD38 and CD19 on CLL cells. Patients with CD200 levels at least 1.4-foldabove the levels found on normal B cells can be selected for treatmentwith anti-CD200 antibodies. As another example, tissue samples from apatient may be stained with anti-CD200 antibody to determine theexpression of CD200 in the patient's malignant and normal cells.

In another example of using the present anti-CD200 antibodies as adiagnostic or prognostic tool, biopsies from patients with malignanciesare obtained and expression of CD200 is determined by FACS analysisusing anti-CD200 antibodies or by immunohistochemistry using anti-CD200.If tumor cells express CD200 at levels that are at least 1.4-fold highercompared to corresponding normal tissue, cancer patients are selectedfor immunomodulatory therapy (including but not limited to a therapycomprising anti-CD200 therapy). For cancer derived from cells thatnormally do not express CD200, any detectable CD200 on cancer biopsiesindicates potential usefulness of anti-CD200 therapy. Immunomodulatorytherapy can be anti-CD200 therapy, but can also be any other therapyaffecting the patient's immune system. Examples of suitableimmunomodulatory therapies include the administration of agents thatblock negative regulation of T cells or antigen presenting cells (e.g.,anti-CTLA4, anti-PD-L1, anti-PDL-2, anti-PD-1) or the administration ofagents that enhance positive co-stimulation of T cells (e.g., anti-CD40or anti 4-1BB). Furthermore, immunomodulatory therapy could be cancervaccines such as heteroclitic peptides or tumor cell peptides thatgenerate cytotoxic T cells or dendritic cells loaded with tumor cells,or the administration of agents that increase NK cell number or T-cellactivity (e.g., anti-CD200 antibodies alone or in combination withinhibitors such as IMiDs, thalidomide, or thalidomide analogs), or theadministration of agents that deplete regulatory T cells (e.g.anti-CD200 antibodies alone or in combination with ONTAK), orplasmacytoid dendritic cells. Combination with agents increasing T cellor dendritic cell migration is also advantageous, such as e.g. any agentblocking SPARC. Furthermore, immunomodulatory therapy could be cancervaccines such as dendritic cells loaded with tumor cells, patientderived exosomes tumor RNA or tumor DNA, tumor protein or tumorpeptides, patient derived heat-shocked proteins (hsp's), hsp's loadedwith tumor antigens or general adjuvants stimulating the immune systemat various levels such as CpG, Luivac, Biostim, Ribominyl, Imudon,Bronchovaxom or any other compound activating receptors of the innateimmune system (e.g., toll like receptors). Also, therapy could includetreatment with cytokines such as IL-2, GM-CSF and IFN-gamma. Combinationwith agents restoring compromised activity of dendritic cells in thetumor environment such as e.g. MAP kinase inhibitors are alsocontemplated.

In one embodiment, the present antibodies also may be utilized to detectcancerous cells in vivo. Detection in vivo is achieved by labeling theantibody, administering the labeled antibody to a subject, and thenimaging the subject. Examples of labels useful for diagnostic imaging inaccordance with the present disclosure are radiolabels such as ¹³¹I,¹¹¹In, ¹²³I, ^(99m)Tc, ³²P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labelssuch as fluorescein and rhodamine, nuclear magnetic resonance activelabels, positron emitting isotopes detectable by a positron emissiontomography (“PET”) scanner, chemiluminescers such as luciferin, andenzymatic markers such as peroxidase or phosphatase. Short-rangeradiation emitters, such as isotopes detectable by short-range detectorprobes, such as a transrectal probe, can also be employed. The antibodycan be labeled with such reagents using techniques known in the art. Forexample, see Wensel and Meares, Radioimmunoimaging andRadioimmunotherapy, Elsevier, N.Y. (1983), which is hereby incorporatedby reference, for techniques relating to the radiolabeling ofantibodies. See also, D. Colcher et al., “Use of Monoclonal Antibodiesas Radiopharmaceuticals for the Localization of Human CarcinomaXenografts in Athymic Mice”, Meth. Enzymol. 121: 802-816 (1986), whichis hereby incorporated by reference.

A radiolabeled antibody in accordance with this disclosure can be usedfor in vitro diagnostic tests. The specific activity of an antibody,binding portion thereof, probe, or ligand, depends upon the half-life,the isotopic purity of the radioactive label, and how the label isincorporated into the biological agent. In immunoassay tests, the higherthe specific activity, in general, the better the sensitivity.Procedures for labeling antibodies with the radioactive isotopes aregenerally known in the art.

The radiolabeled antibody can be administered to a patient where it islocalized to cancer cells bearing the antigen with which the antibodyreacts, and is detected or “imaged” in vivo using known techniques suchas radionuclear scanning using e.g., a gamma camera or emissiontomography. See e.g., A. R. Bradwell et al., “Developments in AntibodyImaging”, Monoclonal Antibodies for Cancer Detection and Therapy, R. W.Baldwin et al., (eds.), pp. 65-85 (Academic Press 1985), which is herebyincorporated by reference. Alternatively, a positron emission transaxialtomography scanner, such as designated Pet VI located at BrookhavenNational Laboratory, can be used where the radiolabel emits positrons(e.g., ¹¹C, ¹⁸F, ¹⁵O, and ¹³N).

Fluorophore and chromophore labeled biological agents can be preparedfrom standard moieties known in the art. Since antibodies and otherproteins absorb light having wavelengths up to about 310 nm, thefluorescent moieties should be selected to have substantial absorptionat wavelengths above 310 nm and preferably above 400 nm. A variety ofsuitable fluorescers and chromophores are described by Stryer, Science,162:526 (1968) and Brand, L. et al., Annual Review of Biochemistry,41:843-868 (1972), which are hereby incorporated by reference. Theantibodies can be labeled with fluorescent chromophore groups byconventional procedures such as those disclosed in U.S. Pat. Nos.3,940,475, 4,289,747, and 4,376,110, which are hereby incorporated byreference.

In another embodiment in accordance with the present disclosure, methodsare provided for monitoring the progress and/or effectiveness of atherapeutic treatment. The method involves administering animmunomodulatory therapy and determining CD200 levels in a subject atleast twice to determine the effectiveness of the therapy. For example,pre-treatment levels of CD200 can be ascertained and, after at least oneadministration of the therapy, levels of CD200 can again be determined.A decrease in CD200 levels is indicative of an effective treatment.Measurement of CD200 levels can be used by the practitioner as a guidefor increasing dosage amount or frequency of the therapy. It should ofcourse be understood that CD200 levels can be directly monitored or,alternatively, any marker that correlates with CD200 can be monitored.Other methods to determine the effectiveness of this therapy include butare not limited to detection of cancer cells, total lymphocyte count,lymph node size, number of regulatory T cells, cytokine profiles in theserum or intracellular, or secretion of cytokines by T or B cells asmeasured by ELISPOT.

C. Other CD200 Antagonists

The CD200 antagonists and polypeptides and/or antibodies utilized in thepresent disclosure are especially indicated for diagnostic andtherapeutic applications as described herein. Accordingly CD200antagonists and anti-CD200 antibodies and variants thereof may be usedin therapies, including combination therapies, in the diagnosis andprognosis of disease, as well as in the monitoring of diseaseprogression.

In the therapeutic embodiments of the present disclosure, bispecificantibodies are contemplated. Bispecific antibodies are monoclonal,preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. In the present case,one of the binding specificities is for the CD200 antigen on a cell(such as, e.g., a cancer cell or immune cell), the other one is for anyother antigen, and preferably for a cell-surface protein or receptor orreceptor subunit.

Methods for making bispecific antibodies are within the purview of thoseskilled in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the co-expression of twoimmunoglobulin heavy-chain/light-chain pairs, where the two heavy chainshave different specificities (Milstein and Cuello, Nature, 305:537-539(1983)). Antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) can be fused toimmunoglobulin constant domain sequences. The fusion preferably is withan immunoglobulin heavy-chain constant domain, including at least partof the hinge, CH2, and CH3 regions. DNAs encoding the immunoglobulinheavy-chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. For further details of illustrative currentlyknown methods for generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986); WO 96/27011;Brennan et al., Science 229:81 (1985); Shalaby et al., J. Exp. Med.175:217-225 (1992); Kostelny et al., J. Immunol. 148(5):1547-1553(1992); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448(1993); and Gruber et al., J. Immunol. 152:5368 (1994); and Tutt et al.,J. Immunol. 147:60 (1991). Bispecific antibodies also includecross-linked or heteroconjugate antibodies. Heteroconjugate antibodiesmay be made using any convenient cross-linking methods. Suitablecross-linking agents are well known in the art, and are disclosed inU.S. Pat. No. 4,676,980, along with a number of cross-linkingtechniques.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins may be linkedto the Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers may be reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).Alternatively, the antibodies can be “linear antibodies” as described inZapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

D. Modes of Administration and Formulations

The route of antibody administration of the antibodies of the presentdisclosure (whether the pure antibody, a labeled antibody, an antibodyfused to a toxin, etc.) is in accord with known methods, e.g., injectionor infusion by intravenous, intraperitoneal, intracerebral,intramuscular, subcutaneous, intraocular, intraarterial, intrathecal,inhalation or intralesional routes, or by sustained release systems. Theantibody is preferably administered continuously by infusion or by bolusinjection. One may administer the antibodies in a local or systemicmanner.

The present antibodies may be prepared in a mixture with apharmaceutically acceptable carrier. Techniques for formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., latest edition. This therapeutic composition can be administeredintravenously or through the nose or lung, preferably as a liquid orpowder aerosol (lyophilized). The composition may also be administeredparenterally or subcutaneously as desired. When administeredsystemically, the therapeutic composition should be sterile,substantially pyrogen-free and in a parenterally acceptable solutionhaving due regard for pH, isotonicity, and stability. For example, apharmaceutical preparation is substantially free of pyrogenic materialsso as to be suitable for administration as a human therapeutic. Theseconditions are known to those skilled in the art.

Pharmaceutical compositions suitable for use include compositionswherein one or more of the present antibodies are contained in an amounteffective to achieve their intended purpose. More specifically, atherapeutically effective amount means an amount of antibody effectiveto prevent, alleviate or ameliorate symptoms of disease or prolong thesurvival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art, especially in light of the detailed disclosureprovided herein. Therapeutically effective dosages may be determined byusing in vitro and in vivo methods.

While the above disclosure has been directed to antibodies, in someembodiments polypeptides derived from such antibodies can be utilized inaccordance with the present disclosure.

EXEMPLIFICATION

Mouse Model and CD200+ Cell Construction

Raji/PBL Model

NOD.CB17-Prkdc<scid> mice (Jackson Laboratory) were injected with 200 μlRPMI containing 4×10⁶ RAJI cells (ATCC) s.c. along with 0, 1, 5 or 10million PBLs. Nine or ten mice were included per group. PBLs wereisolated from 250 ml whole blood on a histopaque gradient followed byred blood cell lysis using 0.9% ammonium chloride. Tumor growth wasmonitored three times a week by measuring length and width with acaliper. Tumor volume was calculated based on length×width×width/2.

Differences between the groups that were injected with PBLs compared tothe group that received tumor cells only were analyzed by 2-tailedunpaired Student's t-test. Significant differences were observed in thegroups that received 5 or 10 million PBLs, but not in the group thatreceived 1 million PBLs from Day 32 on.

Namalwa PBL Model

NOD.CB17-Prkdc<scid> mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected with 200 μl RPMI containing 4×10⁶ Namalwa cells (ATCC) s.c.along with 0, 2 or 10 million PBLs. 9-10 mice were included per group.PBLs were isolated from 250 ml whole blood on a histopaque gradientfollowed by red blood cell lysis using 0.9% ammonium chloride. Tumorgrowth was monitored three times a week by measuring length and widthwith a caliper. Tumor volume was calculated based onlength×width×width/2.

Creation of Stable CD200-Expressing Cell Lines

Stable CD200-expressing Raji and Namalwa cell lines were generated usingthe Virapower Lentiviral Expression System (Invitrogen, Carlsbad,Calif.). A CD200 cDNA was isolated from primary CLL cells by RT-PCRusing forward primer 5′-GACAAGCTTGCAAGGATGGAGAGGCTGGTGA-3′ (SEQ ID NO:34) and reverse primer 5′-GACGGATCCGCCCCTTTTCCTCCTGCTTTTCTC-3′ (SEQ IDNO: 35). The PCR product was cloned into the Gateway entry vectorpCR8/GW/TOPO-TA and individual clones were sequenced. Clones with thecorrect sequence were recombined in both the sense and antisenseorientations into the lentiviral vectors pLenti6/V5/DEST andpLenti6/UbC/V5/DEST using Gateway technology (Invitrogen, Carlsbad,Calif.). The primary difference between these two vectors is thepromoter used to drive CD200 expression: pLenti6/V5/DEST contains thehuman CMV immediate early promoter, whereas pLenti6/UbC/V5/DEST containsthe human ubiquitin C promoter.

High-titer, VSV-G pseudotyped lentiviral stocks were produced bytransient cotransfection of 293-FT cells as recommended by themanufacturer. Raji or Namalwa cells were transduced by resuspending 10⁶cells in 1 ml of growth medium containing 12 μg/ml Polybrene and adding1 ml of lentiviral stock. After incubating the cells overnight at 37°C., the medium containing virus was removed and replaced with 4 ml offresh medium. Two days later, the infected cells were analyzed for CD200expression by flow cytometry. In all experiments, ≧70% of the cells wereCD200⁺, whereas CD200 was undetectable in the parental cell lines and incells transduced with the negative control (antisense CD200) viruses.

To isolate clonal cell lines that overexpress CD200, the infected cellswere selected with blasticidin for 13 days. The concentrations ofblasticidin used were 6 μg/ml for Raji cells or 2 μg/ml for Namalwacells. Stable clones were then isolated by limiting dilution of theblasticidin-resistant cells into 96-well plates. Clones were screened in96-well format by flow cytometry using PE-conjugated Mouse Anti-HumanCD200 (clone MRC OX104, Serotec) and a BD FACSCalibur equipped with aHigh Throughput Sampler. After screening a total of 2000 Raji and 2000Namalwa clones, those clones with the highest CD200 expression wereexpanded for further characterization using conventional techniques.

Example 1 Efficacy of Humanized Versions of C2aB7 in the RAJI_CD200/PBLModel

A) To evaluate whether humanized versions of C2aB7 retain their efficacyin in vivo tumor models, chimeric C2aB7 (see U.S. patent applicationpublication number 2005/0129690) and 3 humanized versions (C2aB7V4V1,C2aB7V3V1 and C2aB7V3V2) as well as the negative control antibodyalxn4100 were tested in the RAJI-CD200/PBL model. RAJI cells transducedwith CD200 were injected s.c. into NOD.CB17-Prkdc<scid> mice, and theability of PBLs to reduce tumor growth in the presence or absence ofchimeric or humanized C2aB7 antibodies or control antibody alxn4100(which does not bind tumor cells) was assessed. Antibodies atconcentrations indicated below were administered initially with thetumor cells, and then twice/week i.v. The following groups were set upwith 10 mice each:

Group 1: 4×10⁶ RAJI_CD200 s.c.

Group 2: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL

Group 3: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+5 mg/kg C2aB7

Group 4: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+20 mg/kg C2aB7V4V1

Group 5: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+5 mg/kg C2aB7V4V1

Group 6: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+20 mg/kg C2aB7V3V1

Group 7: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+5 mg/kg C2aB7V3V1

Group 8: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+5 mg/kg C2aB7V3V2

Group 9: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+20 mg/kg alxn4100

Tumor length and width were measured 3 times a week, and the tumorvolume was calculated by tumor length*width*width/2. FIG. 18 shows that,as expected, CD200 expression on the tumor cells prevented the immunecells from reducing tumor growth. All humanized versions of C2aB7blocked tumor growth by up to 97% at doses of 20 mg/kg. The controlantibody alxn4100 did not affect tumor growth. These data demonstratethat all the humanized antibodies are highly efficacious in blockingtumor growth.

B) Immune Evasion by CD200

Although the human immune system is capable of raising an immuneresponse against many cancer types, that response is insufficient toeradicate the cancer in most patients, possibly due to immune evasionthrough negative regulation of the immune system by the tumor. Weidentified the immune-suppressive molecule CD200 to be upregulated1.5-5.4-fold on chronic lymphocytic leukemia (CLL) cells in all patientsexamined (n=80). Interaction of CD200 with its receptor is known toalter cytokine profiles from Th1 to Th2 in mixed lymphocyte reactions,and to result in the induction of regulatory T cells, which are thoughtto hamper tumor-specific effector T cell immunity. In the present studywe addressed whether CD200 expression on tumor cells plays a role inimmune evasion, thereby preventing elimination of tumor cells by theimmune system in a xenograft hu/SCID mouse model, and whether treatmentwith an antagonistic anti-CD200 antibody affects tumor growth in thismodel.

The human non-Hodgkin's lymphoma cell lines RAH and Namalwa weretransduced with human CD200 and were injected subcutaneously togetherwith human peripheral blood lymphocytes (PBMC) into NOD/SCID mice. Tumorgrowth in mice that received CD200 expressing tumor cells was comparedto tumor growth in mice that received tumor cells not expressing CD200over time. In subsequent experiments, mice were treated with chimeric,or humanized anti-CD200 antibodies (dose range 1 mg/kg to 20 mg/kg) byintravenous injection. Treatment was either started immediately or 7days after tumor cell injection.

PBMCs reduced RAJI or Namalwa tumor growth by up to 75% in the absenceof CD200 expression. In contrast, growth of RAH or Namalwa tumorsexpressing CD200 at levels comparable to CLL was not reduced by PBMCs.Administration of anti-CD200 antibodies at 5 mg/kg resulted in nearlycomplete tumor growth inhibition (1/10 mice developed a small tumor)over the course of the study even when treatment was started 7 daysafter tumor cell injection.

The presence of human CD200 on tumor cells inhibits the ability of humanlymphocytes to eradicate tumor cells. Treatment of CD200-expressingtumors with antagonistic anti-CD200 antibodies inhibits tumor growth,indicating the potential for anti-CD200 therapy as a promising approachfor CLL.

C) Efficacy of C2aB7G1 Versus C2aB7G2/G4 Constructs

To evaluate whether anti-CD200 antibodies without effector function(G2/G4 fusion constructs of C2aB7 as described below) are equally ormore efficacious than the G1 constructs, G1 and G2/G4 versions as wellas the humanized version of C2aB7 (alxn5200) were tested in theRaji_CD200/PBL model. RAJI cells transduced with CD200 as describedabove were injected s.c. into NOD.CB17-Prkdc<scid> mice, and the abilityof PBLs to reduce tumor growth in the presence or absence of chimericanti-CD200 antibodies c2aB7G1 (c2aB7), c2aB7G2/G4 or the humanizedversions hC2aB7V3V1G1 (V3V1), or hC2aB7V3V2G1 (V3V2) or control antibodyalxn4100 was assessed. Antibodies at concentrations indicated below wereadministered initially with the tumor cells and then twice/week i.v. Thefollowing groups were set up with 10 mice each:

Group 1: 4×10⁶ RAJI_CD200 s.c.

Group 2: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL

Group 3: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+20 mg/kg hV3V2-G1

Group 4: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+5 mg/kg alxn 5200

Group 5: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+2.5 mg/kg alxn 5200

Group 6: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+1 mg/kg alxn 5200

Group 7: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+20 mg/kg chC2aB7G2/G4

Group 8: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+5 mg/kg chC2aB7G2/G4

Group 9: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+2.5 mg/kg chC2aB7G2/G4

Group 10: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+1 mg/kg chC2aB7G2/G4

Group 11: 4×10⁶ RAJI_CD200 s.c.+6×10⁶ PBL+20 mg/kg alxn4100

Tumor length and width were measured three times a week, and the tumorvolume was calculated by tumor length*width*width/2. FIG. 19 shows that,as expected, CD200 expression on the tumor cells prevented the immunecells from reducing tumor growth. However, addition of anti-CD200antibodies reduced the tumor volume by up to 100%. While 20 mg/kgC2aB7G1 resulted in growth of small tumors in 6/10 mice, only 1 mousegrew tumors in the group treated with 20 mg/kg C2aB7G2/G4, suggestingthat the G2/G4 version might result in better or at least equal efficacyas the G1 version. All anti-CD200 antibodies, including the humanizedversions, completely blocked tumor growth at 5 mg/kg. Treatment with thecontrol antibody did not reduce the tumor growth. These data prove thatthe G2/G4 version of C2aB7 is highly efficacious in blocking tumorgrowth of CD200 expressing tumors. These data further confirm that thehumanized versions of C2aB7 are highly efficacious in blocking tumorgrowth in this model.

D) Generation of G2/G4 Construct

Plasmids were altered in two steps, first replacing the IgG1 region froman Age I site in the human CH1 region through the stop codon to a BamH Isite located after the SV40 poly A signal. C2aB7-6 and cC7 G2G4 (L-SIGNantibody) were digested with Age I and BamH I, and C2aB7-6 was treatedwith CIP. A 10,315 bp fragment from C2AB7-6 and a 1752 bp fragment fromcC7 G2G4 were purified by electrophoresis and gel extraction. Thesefragments were ligated, electroporated into XL1 Blue E. coli, and platedon LB/carb/gluc plates. Colonies were grown in solution and DNA wasisolated using Qiagen miniprep columns. The presence of the IgG2G4 AgeI/BamH I fragment, as opposed to the IgG1 fragment, was determined byPvu II digestion which results in the presence of two bands of 267 and1152 bp as opposed to one band of 1419 bp. Clone 21 was selected forfurther use.

The remainder of the CH1 region from the end of the variable region tothe Age I site was generated in an IgG2/G4 format by using overlap PCR.The PCR fragment containing the beginning of the CH1 region through theAge I site had previously been generated in the production of plasmidcC7 G2G4. Primers C7 mhHF (TCCTCAGCCTCCACCAAGGGCC, SEQ ID NO:1) and RevAge Pri (GGGCGCCTGAGTTCCACGAC, SEQ ID NO: 2) were used in a PCR reactionwith G2G4 63L1D as template to generate a 142 bp fragment. Primers C2aB7rev (GGCCCTTGGTGGAGGCTGAGGAAACTGTGAGAGTGGTGC, SEQ ID NO: 3) and lacpri(GCTCCCGGCTCGTATGTTGTGT, SEQ ID NO: 4) were used with Fab C2aB7 astemplate to generate the murine heavy chain variable region (andupstream material) in a fragment of about 1250 bp. These fragments werepurified by electrophoresis and gel extraction and were used in overlapPCR with the primers Rev Age Pri (GGGCGCCTGAGTTCCACGAC, SEQ ID NO: 2)and LeadVHpAX (ATATGAAATATCTGCTGCCGACCG, SEQ ID NO: 5) to generate a 558bp fragment that was purified on a PCR purification column. This 558 bpfragment and clone 21 were digested with Xho I and Age I to generate a458 bp fragment that was purified by electrophoresis and gel extraction.Clone 21 was also digested with Xho I and Age I, treated with CIP, andan 11.6 kb fragment was purified by electrophoresis and gel extraction.These fragments were ligated and electroporated into XL1 Blue E. coliand plated on LB/carb/gluc plates. Clone C2aB7G2G4.11 was seen to havethe expected restriction fragments when digested with Pvu II.

The final construct C2AB7G2G4.11 was sequenced. It was discovered thatthe TAA stop codon of the light chain had been mutated to the sequenceTCA such that an additional 6 amino acids would be added to the carboxyterminus of the light chain. It was found to have been present in theIgG1 version clone C2AB7-6 that was the parent for C2AB7G2G4.11.Antibodies containing the G2G4 construct are depicted in FIGS. 10, 11,12, 13, and 15.

Example 2 CD200 Expression on Cancer Cells

A. Determination of CD200 Upregulation in CLL Patients

Lymphocytes from 15 CLL patients were stained with FITC-conjugatedanti-CD5 (e-bioscience), APC-conjugated anti-CD19 (e-bioscience) andPE-conjugated anti-CD200 (Serotec). Lymphocytes from healthy donors werestained accordingly. CD200 expression on CD5+CD19+ cells was determined.As shown in FIG. 20, although the level of CD200 expression varied amongCLL patient samples, all CLL samples showed elevated levels (1.6-4-foldrange) higher CD200 expression compared to CD200 expression on normal Bcells. The CLL patients showing elevated levels of CD200 expression areselected for anti-CD200 treatment in accordance with the methodsdescribed herein.

B. FACS Analysis on Cancer Cell Lines

CD200 expression was evaluated by FACS analysis using a panel of NCI60cell lines from melanoma cancer patients, prostate cancer patients,glioblastoma patients, astrocytoma patients, neuroblastoma patients,ovarian cancer patients, lung cancer patients and renal cancer patients.C2aB7 was labeled with Zenon-Alexa488 according to the manufacturer'sinstructions (Invitrogen). One half million to 1 million cells werestained with 1 μg of the labeled antibody for 20 min, followed by a PBSwash. Cell staining was assessed using a FACSCalibur (Becton Dickinson).Staining of antibody-labeled cells was compared with samples thatremained unlabeled and the ratio of stained/unstained was determined. InFIG. 21, a ratio greater than 1 but smaller than 2 is indicated as +/−,a ratio between 2 and 3 is +, between 3 and 10 is ++, >10 is +++. Noneof the cell lines tested for glioblastoma, astrocytoma, prostate or lungcancer expressed CD200, and are not listed below. Four out of 5 testedmelanoma cell lines, 2/2 ovarian cancer cell lines, 2/3 renal celllines, 2/2 neuroblastoma cell lines and 1/3 breast cancer cell linesexpressed CD200 at detectable levels on the cell surface, suggestingthat solid tumors might use CD200 as an immune escape mechanism as well.

C. RT-QPCR on Patient Samples

To verify whether CD200 is upregulated not only on cell lines, but alsoon primary patient samples, RT-QPCR and immunohistochemistry (IHC) wereperformed on primary patient samples. RNA samples from ovarian andmelanoma patients were obtained from Cytomix. cDNA was prepared andsamples were diluted 1:100 and 1:1000 in 10 ng/ml yeast RNA. Sampleswere run for QPCR with CD200 assay Hs00245978_m1 as provided by ABI. For18S normalization, 18S assay (ABI) was run with samples diluted1:10,000. Each dilution was run in duplicate. Ovarian and melanomapatient samples, along with CLL patient samples were normalized to 18S,then fold expression relative to normal PBL was determined. FIG. 22shows CD200 expression on ovarian cancer samples. Serous/serousmetastatic/papillary serous appeared to have the highest expression ofCD200 at approximately 10- to 20-fold higher than normal PBL. CD200expression was relatively low in endometroid, mucinous, and clear cellsamples, all at or below normal ovary expression levels (1-5 fold higherthan normal PBL).

FIG. 23 shows the CD200 expression levels of several melanoma metastasessamples: jejunum, small intestine, lymph node, lung, skin, and brain).Several of these samples are matched normal/tumor, indicated by thenumber (−1 pair or −2 pair). Other additional samples without matchednormals were also run for comparison. Jejunum samples showedsignificantly higher CD200 expression levels than the normal organ, withthe metastatic samples about 4-7-fold higher than normal jejunum.

D. Immunohistochemistry on Primary Patient Samples

IHC was performed on 2 frozen melanoma patient samples (LifeSpan). D1B5and C2aB7 Fab fragments were used for staining. An IgG1 antibody wasused as isotype control. Binding of the primary antibodies was detectedwith an anti-mouse secondary antibody and DAB chromagen.

As shown in FIG. 24, both melanoma samples tested showed strong membranestaining with the anti-CD200 antibodies, but no staining with theisotype control. Normal skin tissue did not show CD200 staining. Thesedata demonstrate that CD200 is not only upregulated on melanoma andovarian cancer cell lines, but also on primary patient samples.

E. Immune Evasion of Melanoma and Ovarian Tumor Cells ThroughUpregulation of the Immunosuppressive Molecule CD200

Immune escape is a critical feature of cancer progression. Tumors canevade the immune system by multiple mechanisms, each a significantbarrier to immunotherapy. Implementing new and more effective forms ofimmunotherapy will require understanding of these processes as well astheir similarities and differences across cancers. We previouslyidentified the immunosuppressive molecule CD200 to be upregulated onchronic lymphocytic leukemia cells. Presence of CD200 downregulates Th1cytokine production required for an effective cytotoxic T cell response.We demonstrated in animal models that CD200 expression by human tumorcells prevents human lymphocytes from rejecting the tumor, and treatmentwith an antagonistic anti-CD200 antibody inhibited tumor growth. In thisstudy, we evaluated whether CD200 upregulation is found on othercancers, and whether CD200 expression on these cancer cells affects theimmune response.

Relative CD200 message levels were quantitated by RT-QPCR in ovarianadenocarcinoma (serous/serous metastatic/papillary serous, endometroid,mucinous, clear cell) and malignant melanoma metastatic patient samples.

Cell surface expression of CD200 was evaluated by IHC in two melanomaand three ovarian carcinoma (serous) patient frozen tissue samples incomparison with normal skin and normal ovaries. CD200 expression on thecell surface of the melanoma cancer cell lines SK-MEL-5, SK-MEL-24 andSK-MEL-28 and the ovarian cancer cell line OV-CAR-3 was assessed by FACSanalysis using a PE-labeled anti-CD200 antibody. The effect of theCD200-expressing cancer cell lines on cytokine profile mixed inlymphocyte reactions were assessed by adding the cells to a culture ofhuman monocyte-derived dendritic cells with allogeneic human T cells.Cytokine production (IL-2 and IFN-γ for Th1, IL4 and IL10 for Th2) wasdetected in the supernatant by ELISA.

Quantitative PCR showed CD200 expression levels in serous ovarianadenocarcinoma samples at up to 20 fold higher than normal PBL, andequal to or up to 4-fold higher than normal ovary. CD200 expression wasat or below normal ovary levels in endometroid, mucinous, and clear cellovarian adenocarcinoma samples. In malignant melanoma metastases to thejejunum, CD200 expression levels appeared to be significantly higherthan normal samples. In malignant melanoma lung metastases, 2/6 showedhigher CD200 expression than normal samples.

IHC showed strong, specific, membrane-associated CD200 staining onmalignant cells of both melanoma patients. The normal skin sample showedfaint staining of endothelial cells. Among three ovarian cancerpatients, one showed strong CD200 staining, one was moderately positive,and one showed subsets of faintly stained tumor cells. In all threecases, the stroma showed strong staining.

CD200 was highly expressed on the cell surface of the melanoma cancercell lines SK-MEL-24 and SK-MEL-28 as well as on the ovarian cancer cellline OV-CAR-3, and moderately expressed on the melanoma cell lineSK-MEL-5. Addition of any of these cell lines to a mixed lymphocytereaction downregulated the production of Th1 cytokines, while cell linesnot expressing CD200 did not, demonstrating a direct correlation.Inclusion of an antagonistic anti-CD200 antibody during the cultureabrogated the effect.

Melanoma and ovarian tumor cells can upregulate CD200, therebypotentially suppressing an effective immune response. Therapy with anantagonistic anti-CD200 might allow the immune system to mount aneffective cytotoxic response against the tumor cells.

F. Effect of CD200-Expressing Cancer Cell Lines on Cytokine Profiles inMixed Lymphocyte Reactions

The capability of cells overexpressing CD200 to shift the cytokineresponse from a TH1 response (IL-2, IFN-γ) to a Th2 response (IL-4,IL-10) was assessed in a mixed lymphocyte reaction. As a source ofCD200-expressing cells, either CD200 transfected cells or cells fromCD200 positive cancer cell lines were used.

Mixed lymphocyte reactions were set up in 24 well plates using 250,000dendritic cells matured from human peripheral monocytes using IL-4,GM-CSF and IFN-γ and 1×10⁶ responder cells. Responder cells were T cellenriched lymphocytes purified from peripheral blood using Ficoll. Tcells were enriched by incubating the cells for 1 hour in tissue cultureflasks and taking the non-adherent cell fraction. 500,000 cells from themelanoma cancer cell lines SK-MEL-1, SK-MEL-24, SK-MEL-28, the ovariancancer cell line OVCAR3 and the non-Hodgkin's lymphoma cell line Namalwaor primary CLL cells as positive control were added to the dendriticcells in the presence or absence of 30 μg/ml anti-CD200 antibody.Supernatants were collected after 48 and 68 hours and analyzed for thepresence of cytokines

Cytokines such as IL-2, IFN-γ, and IL-10 found in the tissue culturesupernatant were quantified using ELISA. Matched capture and detectionantibody pairs for each cytokine were obtained from R+D Systems(Minneapolis, Minn.), and a standard curve for each cytokine wasproduced using recombinant human cytokine. Anti-cytokine captureantibody was coated on the plate in PBS at the optimum concentration.After overnight incubation, the plates were washed and blocked for 1hour with PBS containing 1% BSA and 5% sucrose. After 3 washes with PBScontaining 0.05% Tween, supernatants were added at dilutions of two-foldor ten-fold in PBS containing 1% BSA. Captured cytokines were detectedwith the appropriate biotinylated anti-cytokine antibody followed by theaddition of alkaline phosphatase conjugated streptavidin and SigmaSsubstrate. Color development was assessed with an ELISA plate reader(Molecular Devices).

As shown in FIG. 25A, the presence of cell lines with high CD200expression (MEL-24, MEL-28, OVCAR-3) resulted in down-regulation of Th1cytokines such as IL-2 and IFN-γ. In contrast, addition of MEL-1 (lowCD200 expression) or Namalwa (no CD200 expression) did not affect thecytokine profile. Addition of the anti-CD200 antibody hB7VH3VL2 at 50μg/ml fully restored the Th1 response (FIG. 25B), indicating thatanti-CD200 antibody treatment of melanoma or ovarian cancer patientsmight be therapeutically beneficial.

Example 3 Elimination of Activated T Cells by C2aB7-G1 and itsDerivatives

To evaluate whether anti-CD200 treatment has an effect in a cancer modelusing tumor cells not expressing CD200, Namalwa cells and human PBLswere injected into NOD/SCID mice, and mice were treated as outlinedbelow. In this model, CD200 is only present on immune cells naturallyexpressing CD200 such as B cells and follicular T-helper cells.

Group Design:

10 animals/group

Group 1: 4×10⁶ Namalwa s.c.

Group 2: 4×10⁶ Namalwa s.c.+8×10⁶ PBL

Group 3: 4×10⁶ Namalwa s.c.+8×10⁶ PBL+20 mg/kg hV3V2-G1

Group 4: 4×10⁶ Namalwa s.c.+8×10⁶ PBL+5 mg/kg hV3V2-G1

Group 5: 4×10⁶ Namalwa s.c.+8×10⁶ PBL+2.5 mg/kg hV3V2-G1

Group 6: 4×10⁶ Namalwa s.c.+4×10⁶ PBL

Group 7: 4×10⁶ Namalwa s.c.+8×10⁶ PBL+20 mg/kg chC2aB7-G2G4

Group 8: 4×10⁶ Namalwa s.c.+8×10⁶ PBL+5 mg/kg chC2aB7-G2G4

Group 9: 4×10⁶ Namalwa s.c.+8×10⁶ PBL+2.5 mg/kg chC2aB7-G2G4

Group 10: 4×10⁶ Namalwa s.c.+4×10⁶ PBL+20 mg/kg chC2aB7-G2G4

Group 11: 4×10⁶ Namalwa s.c.+8×10⁶ PBL+20 mg/kg alxn4100 1/10^(th) ofthe dose was included in the injection mixture. Subsequent dosing was2×/week i.v. for 3 weeks.

Tumor length (L) and width (W) were measured 3 times/week and tumorvolumes were calculated by L*W*W/2. FIG. 26 shows that as previouslyestablished, simultaneous injection of human PBLs with Namalwa cellsinhibits tumor growth. No effect of chC2aB7-G2G4 on PBL-mediated tumorgrowth inhibition was observed. In contrast, administration of ALXN5200(hB7VH3VL2-G 1) blocked PBL mediated tumor growth inhibition. In theabsence of CD200 on tumor cells, it appears that anti-CD200 antibodytreatment with an antibody that mediates effector function such as G1constructs, critical effector cells in the PBL population areeliminated. These data suggest that anti-CD200 cancer therapy is lesseffective when an antibody with effector function is being used ascompared to using the antibody without effector function. However,anti-CD200 treatment using a construct with effector function could betherapeutically beneficial in situations where elimination of immunecells is desirable such as in the transplantation setting or autoimmunediseases.

Example 4 T Cell Killing by hB7VH3VL2

To evaluate whether incubation of activated T cells with anti-CD200antibodies containing a constant region mediating effector function(e.g. G1) results in the killing of the T cells, T cells were activatedand killing assays were set up as described below.

A). CD3+ T Cell Isolation

Human peripheral blood lymphocytes (PBLs) were obtained from normalhealthy volunteers by density gradient centrifugation of heparinizedwhole blood using the Accuspin™ System. Fifteen ml of Histopaque-1077(Sigma, St. Louis, Mo.; cat# H8889) was added to each Accuspin tube(Sigma, St. Louis, Mo.; cat# A2055) which was then centrifuged at 1500rpm for 2 minutes so that the Histopaque was allowed to pass through thefrit. Thirty ml of whole blood was layered over the frit and the tubeswere centrifuged for 15 minutes at 2000 rpm at room temperature with nobrake. The PBL interface was collected and mononuclear cells were washedtwice in PBS with 2% heat-inactivated fetal bovine serum (FBS) (AtlasBiologicals, Ft. Collins, Colo.; cat# F-0500-D) with 1200 rpmcentrifugation for 10 minutes. CD3+ T cells were isolated by passageover a HTCC-5 column (R&D Systems) according to the manufacturer'sinstructions. Eluted cells were washed, counted and resuspended in RPMI1640 containing 5% heat-inactivated single donor serum, 2 mML-glutamine, 10 mM Hepes and penicillin/streptomycin.

B. Activation with Plate-Bound mOKT3

Wells of 12-well plates (Falcon) were coated by overnight incubation at4° C. with 10 μg/mL mOKT3 (Orthoclone) diluted in PBS. Residual antibodywas removed and the plates gently rinsed with PBS. Purified CD3+ Tcells, isolated as described above, were added to the plates at a finalconcentration of 2×10⁶/well in RPMI 1640 containing 5% heat-inactivatedsingle donor serum, 2 mM L-glutamine, 10 mM Hepes andpenicillin/streptomycin. Cells were maintained for 72 hours at 37° C. ina humidified incubator containing 5% CO₂.

C. ⁵¹Chromium Labeling of mOKT3-Activated CD3+ Target Cells

At the end of the culture period, mOKT3-activated CD3+ cells wereharvested, washed and resuspended at 10⁷ cells/mL in RPMI 1640 withoutserum. Cells were chromated by the addition of 125 μCi of ⁵¹Chromium(Perkin Elmer, Billerica, Mass.)/10⁶ cells for 2 hours at 37° C. Labeledcells were harvested, washed in RPMI containing 5% heat-inactivatedsingle donor serum and resuspended at a final concentration of 2×10⁵cells/mL in the same medium.

D. Preparation of Autologous NK Effector Cells

Human peripheral blood lymphocytes (PBLs) from the same individual wereobtained as described above by density gradient centrifugation. The PBLinterface was collected and mononuclear cells were washed twice in PBSwith 2% heat-inactivated fetal bovine serum (FBS) (Atlas Biologicals,Ft. Collins, Colo.; cat# F-0500-D) with 1200 rpm centrifugation for 10minutes. CD56+ cells were isolated by positive selection overanti-CD56-conjugated magnetic beads (Miltenyi Biotec, Auburn, Calif.,Cat # 120-000-307) according to the manufacturer's instructions. Elutedcells were washed, counted and resuspended at 1.3×10⁶ cells/mL in RPMI1640 containing 5% heat-inactivated single donor serum, 2 mML-glutamine, 10 mM Hepes and penicillin/streptomycin. Cells wereincubated overnight at 37° C. in a humidified incubator containing 5%CO, at a final concentration of 4×10⁶ cells/well in 3 mL of the samemedium. At the end of the culture period, the cells were harvested,washed, counted and resuspended in serum-free RPMI containing 2 mML-glutamine, 10 mM Hepes, 2×10⁻⁵M 2-mercaptoethanol andpenicillin/streptomycin.

E. ADCC Assay

⁵¹Cr-labelled mOKT3-activated CD3+ target cells prepared as describedabove were distributed in wells of a 96-well plate at 10⁴ cells/well in50 μl. CD56+ effector cells were harvested, washed, counted andresuspended at either 2.5×10⁶ cells/mL (for an effector:target cellratio of 25:1) or 10⁶ cells/mL (for an effector:target cell ratio of10:1) and were distributed (100 μL/well) to wells containing the targetcells. Ten-fold dilutions of anti-CD200 antibodies (V3V2-G1 orV3V2-G2/G4) were added to the effectors and targets at finalconcentrations of 10, 1, 0.1 and 0.01 μg/mL. Assay controls included thefollowing: 1) effectors and targets in the absence of antibody (0 Ab);2) target cells in the absence of effectors (spontaneous lysis) and 3)effectors and targets incubated with 0.2% Tween-80 (maximum release).All cell culture conditions were performed in triplicate. Cells wereincubated at 37° C. for 4 hours in a humidified incubator containing 5%CO₂. At the end of the culture period, the plates were centrifuged topellet the cells and 150 μL of cell supernatant was transferred toscintillation vials and counted in a gamma scintillation counter(Wallac). The results are expressed as percent specific lysis accordingto the following formula:

$\frac{\begin{matrix}{\text{(Mean~~sample~~counts~~per~~minute~~(cpm)} -} \\\text{mean~~spontaneous~~lysis)}\end{matrix}}{{\text{(mean~~maximum~~lysis} - \text{mean~~spontaneous~~lysis)}}\;} \times 100$F. Flow Cytometry

One hundred μl of cell suspensions (mOKT3-activated CD3+ cells orpurified CD56+ NK cells) prepared as described above were distributed towells of a 96-well round bottom plate (Falcon, Franklin Lakes N.J.;cat#353077). Cells were incubated for 30 minutes at 4° C. with theindicated combinations of the following fluorescein isothiocyanate(FITC)-, Phycoerythrin (PE)-, PerCP-Cy5.5-, or allophycocyanin(APC)-conjugated antibodies (all from Becton-Dickinson, San Jose,Calif.); anti-human CD25-FITC (cat#555431); anti-human CD3-APC(cat#555335); anti-human CD200-PE (cat #552475); anti-humanCD8-PerCP-Cy5.5 (cat#341051); anti-human CD4-APC (cat#555349);anti-human CD5-APC (cat#555355) and anti-human CD56-APC (cat#341025).Isotype controls for each labeled antibody were also included. Afterwashing cells twice with FACS buffer (1800 rpm centrifugation for 3minutes), cells were resuspended in 300 μl of PBS (Mediatech, Herndon,Va.; cat#21-031-CV) and analyzed by flow cytometry using a FacsCalibermachine and CellQuest Software (Becton Dickinson, San Jose, Calif.).

As shown in FIG. 27, activated T cells show high CD200 expression ontheir surface. Activated T cells are efficiently killed in the presenceof VH3VL2-G1 but not VH3VL2-G2G4 when NK cells are used as effectorcells (FIG. 28). These data demonstrate that anti-CD200 antibodies witheffector function can eliminate activated T cells. Such an antibodycould be of therapeutic use in the transplantation setting or for thetreatment of autoimmune diseases.

In addition to regulatory T cells, plasmacytoid dendritic cells havebeen shown to play a negative immunoregulatory role in human cancer (WeiS, Kryczek I, Zou L, Daniel B, Cheng P, Mottram P, Curiel T, Lange A,Zou W Plasmacytoid dendritic cells induce CD8+ regulatory T cells inhuman ovarian carcinoma. Cancer Res. 2005 Jun. 15; 65(12):5020-6).Combination of a therapy eliminating plasmacytoid dendritic cells withanti-CD200 therapy could therefore be advantageous.

Example 5 CD200 on Plasma Cells

Bone marrow cells from 10 multiple myeloma patients and 3 normal donorswere prepared by first lysing red blood cells using ammonium chloride.Cells were resuspended in FACS buffer and labeled with the followingantibody cocktails:

Kappa-FITC/CD38-PE/CD138-PerCP-Cy5.5

Lambda-FITC/CD38-PE/CD138-PerCP-Cy5.5

Isotype Control-FITC/CD38-PE

CD200-FITC/CD38-PE

Data were collected using a BD FACS Canto and analyzed using BD DiVAsoftware. Expression of CD200 on CD38 bright cells (plasma cells) wasanalyzed. As shown in FIG. 29, a portion of plasma cells expresses CD200at high intensity in normal donors. In multiple myeloma patients, themajority of plasma cells express CD200.

In the multiple myeloma setting, similar to CLL or other cancersexpressing CD200, CD200 expression by the tumor cells might prevent theimmune system from eradicating the tumor cells. Antagonistic anti-CD200therapy might subsequently allow the immune system to eliminate cancercells. Ablative anti-CD200 therapy targeting plasma cells could betherapeutically beneficial in the autoimmune or transplantation setting.

Example 6 CD200 on Viruses

CD200 is also expressed on a number of viruses such as myxoma virusM141R or human herpesvirus 8. Similar to expression of CD200 on tumorcells, CD200 on viruses might prevent the immune system from effectivelyclearing the virus. Treatment with an antagonistic anti-CD200 antibodycould be therapeutically beneficial in an infection with a CD200expressing virus, allowing the immune system to eradicate the virus.Alternatively, an ablative anti-CD200 antibody could be used.

It will be understood that various modifications may be made to theembodiments disclosed herein. For example, as those skilled in the artwill appreciate, the specific sequences described herein can be alteredslightly without necessarily adversely affecting the functionality ofthe polypeptide, antibody or antibody fragment used in bindingOX-2/CD200. For instance, substitutions of single or multiple aminoacids in the antibody sequence can frequently be made without destroyingthe functionality of the antibody or fragment. Thus, it should beunderstood that polypeptides or antibodies having a degree of identitygreater than 70% to the specific antibodies described herein are withinthe scope of this disclosure. In particularly useful embodiments,antibodies having an identity greater than about 80% to the specificantibodies described herein are contemplated. In other usefulembodiments, antibodies having an identity greater than about 90% to thespecific antibodies described herein are contemplated. Therefore, theabove description should not be construed as limiting, but merely asexemplifications of preferred embodiments. Those skilled in the art willenvision other modifications within the scope and spirit of thisdisclosure.

REFERENCES

The following references are incorporated herein by reference to morefully describe the state of the art to which the present inventionpertains. Any inconsistency between these publications below or thoseincorporated by reference above and the present disclosure shall beresolved in favor of the present disclosure.

-   1) Agarwal, et al., (2003). Disregulated expression of the Th2    cytokine gene in patients with intraoral squamous cell carcinoma.    Immunol Invest 32:17-30.-   2) Almasri, N M et al. (1992). Am J Hematol 40 259-263.-   3) Contasta, et al., (2003). Passage from normal mucosa to adenoma    and colon cancer: alteration of normal sCD30 mechanisms regulating    TH1/TH2 cell functions. Cancer Biother Radiopharm 18:549-557.-   4) Gorczynski, et al., (1998). Increased expression of the novel    molecule OX-2 is involved in prolongation of murine renal allograft    survival. Transplantation 65:1106-1114.-   5). Gorczynski, et al., (2001). Evidence of a role for CD200 in    regulation of immune rejection of leukaemic tumour cells in C57BU6    mice. Clin Exp Immunol 126:220-229.-   6) Hainsworth, J D (2000). Oncologist 2000; 5(5):376-84.-   7) Inagawa, et al., (1998). Mechanisms by which chemotherapeutic    agents augment the antitumor effects of tumor necrosis factor:    involvement of the pattern shift of cytokines from Th2 to Th1 in    tumor lesions. Anticancer Res 18:3957-3964.-   8) Ito, et al., (1999). Lung carcinoma: analysis of T helper type 1    and 2 cells and T cytotoxic type 1 and 2 cells by intracellular    cytokine detection with flow cytometry. Cancer 85:2359-2367.-   9) Kiani, et al., (2003). Normal intrinsic Th1/Th2 balance in    patients with chronic phase chronic myeloid leukemia not treated    with interferon-alpha or imatinib. Haematologica 88:754-761.-   10) Lauerova, et al., (2002). Malignant melanoma associates with    Th1/Th2 imbalance that coincides with disease progression and    immunotherapy response. Neoplasma 49:159-166.-   11) Maggio, et al., (2002). Chemokines, cytokines and their    receptors in Hodgkin's lymphoma cell lines and tissues. Ann Oncol 13    Suppl 1:52-56.-   12) Nilsson, K (1992). Burn Cell. 5(1):25-41.-   13) Podhorecka, et al., (2002). T type 1/type 2 subsets balance in    B-cell chronic lymphocytic leukemia—the three-color flow cytometry    analysis. Leuk Res 26:657-660.-   14) Pu, Q Q and Bezwoda, W (2000). Anticancer Res. 20(4):2569-78-   15) Smyth, et al., (2003). Renal cell carcinoma induces    prostaglandin E2 and T-helper type 2 cytokine production in    peripheral blood mononuclear cells. Ann Surg Oncol 10:455-462.-   16) Tatsumi, et al., (2002). Disease-associated bias in T helper    type 1 (Th1)/Th2 CD4(+) T cell responses against MAGE-6 in    HLA-DRB10401(+) patients with renal cell carcinoma or melanoma. J    Exp Med 196:619-628.-   17) Walls A V et al. (1989). Int. J. Cancer 44846-853.-   18) Winter, et al., (2003). Tumour-induced polarization of tumour    vaccine-draining lymph node T cells to a type 1 cytokine profile    predicts inherent strong immunogenicity of the tumour and correlates    with therapeutic efficacy in adoptive transfer studies. Immunology    108:409-419.-   19) Cameron, C. M., J. W. Barrett, L. Liu, A. R. Lucas, and G.    McFadden. 2005. Myxoma virus M141R expresses a viral CD200 (vOX-2)    that is responsible for down-regulation of macrophage and T-cell    activation in vivo. J Virol 79:6052.-   20) Foster-Cuevas, M., G. J. Wright, M. J. Puklavec, M. H. Brown,    and A. N. Barclay. 2004. Human herpesvirus 8 K14 protein mimics    CD200 in down-regulating macrophage activation through CD200    receptor. J Virol 78:7667.-   21) Nicholas, J. 2003. Human herpesvirus-8-encoded signalling    ligands and receptors. J Biomed Sci 10:475.-   22) Shiratori, I., M. Yamaguchi, M. Suzukawa, K. Yamamoto, L. L.    Lanier, T. Saito, and H. Arase. 2005. Down-regulation of basophil    function by human CD200 and human herpesvirus-8 CD200. J Immunol    175:4441.-   23) Voigt, S., G. R. Sandford, G. S. Hayward, and W. H. Burns. 2005.    The English strain of rat cytomegalovirus (CMV) contains a novel    captured CD200 (vOX2) gene and a spliced CC chemokine upstream from    the major immediate-early region: further evidence for a separate    evolutionary lineage from that of rat CMV Maastricht. J Gen Virol    86:263.-   24) Zhang, J., J. Wang, C. Wood, D. Xu, and L. Zhang. 2005. Kaposi's    sarcoma-associated herpesvirus/human herpesvirus 8 replication and    transcription activator regulates viral and cellular genes via    interferon-stimulated response elements. J Virol 79:5640.

1. A pharmaceutical composition comprising a pharmaceutically-acceptablecarrier and an anti-CD200 antibody comprising an altered Fc constantregion, wherein said anti-CD200 antibody inhibits the interactionbetween CD200 and CD200R, and wherein said altered Fc constant regionexhibits decreased effector function relative to the effector functionof the corresponding native Fc constant region.
 2. The pharmaceuticalcomposition of claim 1, wherein the effector function comprises one ormore of the following: (a) antibody-dependent cell-mediated cytotoxicity(ADCC); (b) complement dependent cytotoxicity (CDC); or (c) binding toone or more Fc receptors.
 3. The pharmaceutical composition of claim 1,wherein the anti-CD200 antibody is a murine antibody, a chimericantibody, a humanized antibody, a deimmunized antibody, or a humanantibody.
 4. The pharmaceutical composition of claim 1, wherein thealtered Fc constant region is an altered form of a native constantregion selected from the group consisting of IgG1, IgG2, IgG3, IgG4,IgM, IgA1, IgA2, IgA, IgD, and IgE.
 5. The pharmaceutical composition ofclaim 1, wherein the altered Fc constant region comprises at least oneamino acid substitution, insertion, or deletion, relative to thecorresponding native constant region.
 6. The pharmaceutical compositionof claim 1, wherein: (a) the altered Fc constant region is a G2/G4constant region; or (b) relative to the native Fc constant region, saidaltered Fc constant region comprises: (i) altered glycosylation; (ii) anAla-Ala mutation; (iii) the CH1 and hinge regions of an IgG2 antibody;or (iv) the CH2 and CH3 regions of an IgG4 antibody.
 7. Thepharmaceutical composition of claim 6, wherein the altered glycosylationcomprises one or more of the following: (i) a change in one or moresugar components; (ii) presence of one or more additional sugarcomponents; and (iii) absence of one or more sugar components.
 8. Thepharmaceutical composition of claim 7, wherein said antibody isexpressed in a host cell selected from the group consisting of amammalian cell, a bacterial cell, and a plant cell.
 9. Thepharmaceutical composition of claim 8, wherein the host cell is E. coli.10. The pharmaceutical composition of claim 8, wherein the host cell isa rat-hybridoma cell.
 11. The pharmaceutical composition of claim 8,wherein the host cell is a CHO cell.
 12. The pharmaceutical compositionof claim 6, wherein the G2/G4 constant region comprises the amino acidsequence depicted in any one of SEQ ID NOs: 13, 15, 18, or
 22. 13. Thepharmaceutical composition of claim 1, wherein the anti-CD200 antibodycomprises: (a) a light chain polypeptide comprising: (i) a light chainvariable domain comprising a light chain CDR1 having the amino acidsequence set forth in residues 44 to 51 of SEQ ID NO:47; a light chainCDR2 having the amino acid sequence set forth in residues 70 to 76 ofSEQ ID NO:47; and a light chain CDR3 having the amino acid sequence setforth in residues 109 to 117 of SEQ ID NO:47; (ii) a light chainvariable domain having an amino acid sequence comprising residues 19 to128 of SEQ ID NO:47; or (iii) an amino acid sequence comprising residues19 to 240 of SEQ ID NO:47; and (b) a heavy chain polypeptide comprising:(iv) a heavy chain variable domain comprising: a heavy chain CDR1 havingthe amino acid sequence set forth in residues 46 to 55 of SEQ ID NO:15;a heavy chain CDR2 having the amino acid sequence set forth in residues70 to 86 of SEQ ID NO:15; and a heavy chain CDR3 having the amino acidsequence set forth in residues 119 to 126 of SEQ ID NO:15. (v) a heavychain variable domain having an amino acid sequence comprising residues20 to 137 of SEQ ID NO:15; or (vi) an amino acid sequence comprisingresidues 20 to 463 of SEQ ID NO:15.
 14. The pharmaceutical compositionof claim 1, wherein the anti-CD200 antibody comprises: (a) a light chainpolypeptide comprising: (i) a light chain variable domain comprising: alight chain CDR1 having the amino acid sequence set forth in residues 44to 54 of SEQ ID NO:32; a light chain CDR2 having the amino acid sequenceset forth in residues 70 to 76 of SEQ ID NO:32; and a light chain CDR3having the amino acid sequence set forth in residues 109 to 117 of SEQID NO:32; (ii) a light chain variable domain having an amino acidsequence comprising residues 21 to 127 of SEQ ID NO:32; or (iii) anamino acid sequence comprising residues 21 to 234 of SEQ ID NO:32; and(b) a heavy chain polypeptide comprising: (iv) a heavy chain CDR1 havingthe amino acid sequence set forth in residues 46 to 55 of SEQ ID NO:20;a ‘heavy chain CDR2 having the amino acid sequence set forth in residues70 to 86 of SEQ ID NO:20; and a heavy chain CDR3 having the amino acidsequence set forth in residues 119 to 131 of SEQ ID NO:20; or (v) aheavy chain variable domain having an amino acid sequence comprisingresidues 21 to 142 of SEQ ID NO:20.
 15. The pharmaceutical compositionof claim 1, wherein the anti-CD200 antibody comprises: (a) a light chainpolypeptide comprising: (i) a light chain variable domain comprising: alight chain CDR1 having the amino acid sequence set forth in residues 44to 55 of SEQ ID NO:30; a light chain CDR2 having the amino acid sequenceset forth in residues 71 to 77 of SEQ ID NO:30; and a light chain CDR3having the amino acid sequence set forth in residues 110 to 118 of SEQID NO:30; (ii) a light chain variable domain having an amino acidsequence comprising residues 21 to 128 of SEQ ID NO:30; or (iii) anamino acid sequence comprising residues 21 to 235 of SEQ ID NO:30; and(b) a heavy chain polypeptide comprising: (iv) a heavy chain variabledomain comprising: a heavy chain CDR1 having the amino acid sequence setforth in residues 46 to 55 of SEQ ID NO:18; a heavy chain CDR2 havingthe amino acid sequence set forth in residues 70 to 86 of SEQ ID NO:18;and a heavy chain CDR3 having the amino acid sequence set forth inresidues 119 to 128 of SEQ ID NO:18; (v) a heavy chain variable domainhaving an amino acid sequence comprising residues 21 to 139 of SEQ IDNO:18; or (vi) an amino acid sequence comprising residues 21 to 468 ofSEQ ID NO:18.
 16. The pharmaceutical composition of claim 1, wherein theanti-CD200 antibody comprises: (a) a light chain polypeptide comprising:(i) a light chain variable domain having an amino acid sequencecomprising residues 23 to 129 of SEQ ID NO:28; or (ii) an amino acidsequence comprising residues 23 to 236 of SEQ ID NO:28; and (b) a heavychain polypeptide comprising: (iii) a heavy chain variable domain havingan amino acid sequence comprising residues 20 to 136 of SEQ ID NO:13; or(iv) an amino acid sequence comprising residues 20 to 462 of SEQ IDNO:13.
 17. The pharmaceutical composition of claim 1, wherein thealtered Fc constant region has 0 to 20% of the FcR binding of thecorresponding native Fc constant region.
 18. The pharmaceuticalcomposition of claim 1, wherein the altered Fc constant region has noeffector function as compared to the corresponding native Fc constantregion.
 19. The pharmaceutical composition of claim 1, wherein thealtered Fc constant region has reduced or no ADCC or CDC activity ascompared to the corresponding native Fc constant region.
 20. Ananti-CD200 antibody comprising an altered Fc constant region, whereinsaid anti-CD200 antibody inhibits the interaction between CD200 andCD200R, wherein said altered Fc constant region exhibits decreasedeffector function relative to the effector function of the correspondingnative Fc constant region, and wherein the anti-CD200 antibodycomprises: (I.) (a) a light chain polypeptide comprising: (i) a lightchain variable domain comprising a light chain CDR1 having the aminoacid sequence set forth in residues 44 to 51 of SEQ ID NO:47; a lightchain CDR2 having the amino acid sequence set forth in residues 70 to 76of SEQ ID NO:47; and a light chain CDR3 having the amino acid sequenceset forth in residues 109 to 117 of SEQ ID NO:47; (ii) a light chainvariable domain having an amino acid sequence comprising residues 19 to128 of SEQ ID NO:47; or (iii) an amino acid sequence comprising residues19 to 240 of SEQ ID NO:47; and (b) a heavy chain polypeptide comprising:(iv) a heavy chain variable domain comprising: a heavy chain CDR1 havingthe amino acid sequence set forth in residues 46 to 55 of SEQ ID NO:15;a heavy chain CDR2 having the amino acid sequence set forth in residues70 to 86 of SEQ ID NO:15; and a heavy chain CDR3 having the amino acidsequence set forth in residues 119 to 126 of SEQ ID NO:15; (v) a heavychain variable domain having an amino acid sequence comprising residues20 to 137 of SEQ ID NO:15; or (vi) an amino acid sequence comprisingresidues 20 to 463 of SEQ ID NO:15; (II.) (a) a light chain polypeptidecomprising: (i) a light chain variable domain comprising: a light chainCDR1 having the amino acid sequence set forth in residues 44 to 54 ofSEQ ID NO:32; a light chain CDR2 having the amino acid sequence setforth in residues 70 to 76 of SEQ ID NO:32; and a light chain CDR3having the amino acid sequence set forth in residues 109 to 117 of SEQID NO:32; (ii) a light chain variable domain having an amino acidsequence comprising residues 21 to 127 of SEQ ID NO:32; or (iii) anamino acid sequence comprising residues 21 to 234 of SEQ ID NO:32; and(b) a heavy chain polypeptide comprising: (iv) a heavy chain CDR1 havingthe amino acid sequence set forth in residues 46 to 55 of SEQ ID NO:20;a heavy chain CDR2 having the amino acid sequence set forth in residues70 to 86 of SEQ ID NO:20; and a heavy chain CDR3 having the amino acidsequence set forth in residues 119 to 131 of SEQ ID NO:20; or (v) aheavy chain variable domain having an amino acid sequence comprisingresidues 21 to 142 of SEQ ID NO:20; (III.) (a) a light chain polypeptidecomprising: (i) a light chain variable domain comprising: a light chainCDR1 having the amino acid sequence set forth in residues 44 to 55 ofSEQ ID NO:30; a light chain CDR2 having the amino acid sequence setforth in residues 71 to 77 of SEQ ID NO:30; and a light chain CDR3having the amino acid sequence set forth in residues 110 to 118 of SEQID NO:30; (ii) a light chain variable domain having an amino acidsequence comprising residues 21 to 128 of SEQ ID NO:30; or (iii) anamino acid sequence comprising residues 21 to 235 of SEQ ID NO:30; and(b) a heavy chain polypeptide comprising: (iv) a heavy chain variabledomain comprising: a heavy chain CDR1 having the amino acid sequence setforth in residues 46 to 55 of SEQ ID NO:18; a heavy chain CDR2 havingthe amino acid sequence set forth in residues 70 to 86 of SEQ ID NO:18;and a heavy chain CDR3 having the amino acid sequence set forth inresidues 119 to 128 of SEQ ID NO:18; (v) a heavy chain variable domainhaving an amino acid sequence comprising residues 21 to 139 of SEQ IDNO:18; or (vi) an amino acid sequence comprising residues 21 to 468 ofSEQ ID NO:18; or (IV.) (a) a light chain polypeptide comprising: (i) alight chain variable domain having an amino acid sequence comprisingresidues 23 to 129 of SEQ ID NO:28; or (ii) an amino acid sequencecomprising residues 23 to 236 of SEQ ID NO:28; and (b) a heavy chainpolypeptide comprising: (iii) a heavy chain variable domain having anamino acid sequence comprising residues 20 to 136 of SEQ ID NO:13; or(iv) an amino acid sequence comprising residues 20 to 462 of SEQ IDNO:13.
 21. The anti-CD200 antibody of claim 20, wherein the effectorfunction comprises one or more of the following: (a) antibody-dependentcell-mediated cytotoxicity (ADCC); (b) complement dependent cytotoxicity(CDC); or (c) binding to one or more Fc receptors.
 22. The anti-CD200antibody of claim 20, wherein the anti-CD200 antibody is a murineantibody, a chimeric antibody, a humanized antibody, a deimmunizedantibody, or a human antibody.
 23. The anti-CD200 antibody of claim 20,wherein the altered Fc constant region is an altered form of a native Fcconstant region selected from the group consisting of IgG1, IgG2, IgG3,IgG4, IgM, IgA1, IgA2, IgA, IgD, and IgE.
 24. The anti-CD200 antibody ofclaim 20, wherein the altered Fc constant region comprises at least oneamino acid substitution, insertion, or deletion, relative to thecorresponding native constant region.
 25. The anti-CD200 antibody ofclaim 20, wherein: (a) the altered Fc constant region is a G2/G4constant region; or (b) relative to the native Fc constant region, saidaltered Fc constant region comprises: (i) altered glycosylation; (ii) anAla-Ala mutation; (iii) the CH1 and hinge regions of an IgG2 antibody;or (iv) the CH2 and CH3 regions of an IgG4 antibody.
 26. An anti-CD200antibody comprising an altered Fc constant region, wherein saidanti-CD200 antibody inhibits the interaction between CD200 and CD200R,wherein said altered Fc constant region exhibits decreased effectorfunction relative to the effector function of the corresponding nativeFc constant region, wherein said altered Fc constant region is a G2/G4constant region, and wherein said G2/G4 constant region comprises theamino acid sequence depicted in any one of SEQ ID NOs: 13, 15, 18, or22.